Mélina Bailly
email: melina.bailly@uca.fr

Léonard Féasson
email: leonard.feasson@univ-st-etienne.fr.

Bruno Pereira
email: bpereira@chu-clermontferrand.fr.

Amandine Boileau
email: amandine.boileau@uca.fr.

Christophe Hourdé
email: christophe.hourde@univ-smb.fr.

Natacha Germain
email: natacha.germain@chu-st-etienne.fr

Bogdan Galusca
email: bogdan.galusca@chu-st-etienne.fr.

Daniel Courteix
email: daniel.courteix@uca.fr

David Thivel
email: david.thivel@uca.fr

Julien Verney
email: julien.verney@uca.fr

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle

Keywords: Capillary supply, Immunohistochemistry, Combined staining method, Muscle fiber type, CD31

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Introduction

Human muscle is highly heterogeneous and composed of different types of fibers: slow oxidative fibers (type I fibers), fast oxidative glycolytic fibers (type IIA fibers) and fast glycolytic fibers (type IIX fibers) [START_REF] Schiaffino | Fiber types in mammalian skeletal muscles[END_REF]. This classification of muscle fiber types relied on different methods.

Since the '70s, it has been assessed using the myofibrillar adenosinetriphosphatase (mATPase) method [START_REF] Brooke | Muscle fiber types: how many and what kind?[END_REF][START_REF] Johnson | Data on the distribution of fibre types in thirty-six human muscles. An autopsy study[END_REF][START_REF] Essén | Metabolic characteristics of fibre types in human skeletal muscle[END_REF][START_REF] Denis | Effects of endurance training on capillary supply of human skeletal muscle on two age groups (20 and 60 years)[END_REF][START_REF] Proctor | Oxidative capacity of human muscle fiber types: effects of age and training status[END_REF][START_REF] Gavin | Aging and the Skeletal Muscle Angiogenic Response to Exercise in Women[END_REF], based on mATPase sensitivity to pH. In the '90s, the development of the first monoclonal antibodies directed against myosin heavy chains (MHC) gradually replaced mATPase staining [START_REF] Schiaffino | Three myosin heavy chain isoforms in type 2 skeletal muscle fibres[END_REF][START_REF] Gorza | Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies[END_REF][START_REF] Kadi | Structural changes in male trapezius muscle with work-related myalgia[END_REF]. In 2012, Bloemberg & Quadrilatero performed a fine muscle fiber typing (type I, IIA, and IIX fibers) on a single muscle section [START_REF] Bloemberg | Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis[END_REF]. Furthermore, capillary supply is also a major determinant of muscle phenotype, and techniques of capillaries identification have also progressed a lot. First techniques to identify capillaries with the combination of amylase and periodic acid schiff staining [START_REF] Andersen | Capillary density in skeletal muscle of man[END_REF]) have been progressively superseded by the use of the Ulex europaeus agglutinin 1 (UEA 1) lectin, anti-collagen type IV, anti-von Willebrand factor [START_REF] Parsons | Capillarity of elite cross-country skiers: a lectin (Ulex europaeus I) marker[END_REF][START_REF] Madsen | Capillary density measurements in skeletal muscle using immunohistochemical staining with anti-collagen type IV antibodies[END_REF][START_REF] Qu | Visualisation of capillaries in human skeletal muscle[END_REF], and then by the CD31 antibody [START_REF] Charles | Effect of endurance training on muscle microvascular filtration capacity and vascular bed morphometry in the elderly[END_REF][START_REF] Merlet | Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients[END_REF]) which recognizes the platelet endothelial cell adhesion molecule-1 (PECAM-1)a transmembrane glycoprotein expressed by vascular endothelial cells. Capillary supply is commonly assessed through global indexes independent of the fiber type (such as the capillary density (CD) or the capillary to fiber ratio (C/F)) but is also frequently assessed through local indexes in a fiber-type specific manner (such as capillary contacts per fiber (CC), CC per fiber cross-sectional area (CAFA) or capillary to fiber perimeter exchange index (CFPE)) [START_REF] Hepple | Quantitating the capillary supply and the response to resistance training in older men[END_REF][START_REF] Hepple | A new measurement of tissue capillarity: the capillary-to-fibre perimeter exchange index[END_REF][START_REF] Harris | The influence of endurance and resistance exercise on muscle capillarization in the elderly: a review[END_REF][START_REF] Charles | Effect of endurance training on muscle microvascular filtration capacity and vascular bed morphometry in the elderly[END_REF]). The capillary network can even be further analyzed using morphometric indexes such as the capillary tortuosity index (CapTor) [START_REF] Vincent | Remodeling of skeletal muscle microvasculature in sickle cell trait and alpha-thalassemia[END_REF][START_REF] Ravelojaona | Evidence for a profound remodeling of skeletal muscle and its microvasculature in sickle cell anemia[END_REF] or the ratio between the length of contact with the muscle fiber (LC) to the perimeter of muscle fiber (LC/PF) [START_REF] Charifi | Enhancement of microvessel tortuosity in the vastus lateralis muscle of old men in response to endurance training[END_REF][START_REF] Merlet | Beneficial effects of endurance exercise training on skeletal muscle microvasculature in sickle cell disease patients[END_REF]. To date, analyses of local capillary indexes require serial cross-sectioning, which present several limitations. First, serial cross-sectioning is tissue-consuming while human tissues are difficult to obtain and most of the time collected in reduced quantity. Serial cross-sectioning analyses are also time-consuming as the same fibers must be clearly identified among hundreds of others over several cross-sections. This also presents risks of bias due to the changes of fiber types and capillaries along muscle fibers. From these limitations, the need for simultaneous identification of fiber types and capillaries had already been raised more than thirty years ago [START_REF] Rosenblatt | A histochemical method for the simultaneous demonstration of capillaries and fiber type in skeletal muscle[END_REF][START_REF] Paljärvi | Histochemical method for simultaneous fiber typing and demonstration of capillaries in skeletal muscle[END_REF][START_REF] Eržen | Simultaneous histochemical demonstration of capillaries and muscle fibre types[END_REF], even suggesting that immunohistochemical techniques could provide further possibilities. In more recent publications, new immunofluorescence techniques succeeded to stain capillaries, type I fibers, type II fibers, and laminin (a protein component of basal lamina) on a single muscle section [START_REF] Snijders | Muscle fibre capillarization is a critical factor in muscle fibre hypertrophy during resistance exercise training in older men[END_REF][START_REF] Snijders | Muscle fiber capillarization as determining factor on indices of insulin sensitivity in humans[END_REF][START_REF] Nederveen | Skeletal muscle satellite cells are located at a closer proximity to capillaries in healthy young compared with older men[END_REF][START_REF] Nederveen | The influence of capillarization on satellite cell pool expansion and activation following exercise-induced muscle damage in healthy young men[END_REF][START_REF] Tan | Skeletal muscle fiber-type-specific changes in markers of capillary and mitochondrial content after low-volume interval training in overweight women[END_REF][START_REF] Moro | Low skeletal muscle capillarization limits muscle adaptation to resistance exercise training in older adults[END_REF]). However, these techniques did not distinguish type IIA from type IIX muscle fibers. Type IIX fibers present a specific phenotypefor review see [START_REF] Schiaffino | Fiber types in mammalian skeletal muscles[END_REF]. For instance, they present a lower capillarization than type IIA fibers [START_REF] Andersen | Capillary density in skeletal muscle of man[END_REF][START_REF] Larsson | Muscle fiber characteristics in postmenopausal women with normal or impaired glucose tolerance[END_REF][START_REF] Campos | Muscular adaptations in response to three different resistance-training regimens: specificity of repetition maximum training zones[END_REF][START_REF] Gavin | Aging and the Skeletal Muscle Angiogenic Response to Exercise in Women[END_REF], and in response to training their proportion decreases, contrary to the proportion of type IIA fibers, which sometimes increases [START_REF] Gavin | Aging and the Skeletal Muscle Angiogenic Response to Exercise in Women[END_REF][START_REF] Kosek | Efficacy of 3 days/wk resistance training on myofiber hypertrophy and myogenic mechanisms in young vs. older adults[END_REF][START_REF] Mohr | Long-term adaptation to electrically induced cycle training in severe spinal cord injured individuals[END_REF]). Therefore, in training studies, it is of high interest to separately analyze type IIA from type IIX fibers. To the best of our knowledge, only one publication reported the staining of the three muscle fiber types and basal lamina using immunohistochemical techniques along with the staining of capillaries in humans (Al-Shammari et al. 2019)but using UEA 1 instead of CD31 antibody, which is known to be one of the most specific and sensitive markers of endothelial cells [START_REF] Duscha | Capillary density of skeletal muscle: A contributing mechanism for exercise intolerance in class II-III chronic heart failure independent of other peripheral alterations[END_REF]. Thus, we aimed to propose accurate and reliable immunohistochemical methods identifying basal lamina, capillaries with CD31 antibody, as well as type I, IIA and IIX muscle fibers, on a single muscle section.

Materials and methods

The present study used muscle samples from a previous protocol [START_REF] Galusca | Reduced fibre size, capillary supply and mitochondrial activity in constitutional thinness' skeletal muscle[END_REF]) that was conducted in accordance with the Helsinki Declaration and approved by the local research and ethics committee of Saint-Étienne -France. All subjects gave written informed consent prior to inclusion in the study and the protocol was registered at ClinicalTrials.gov as NCT01224561.

Muscle biopsies

Biopsies from the vastus lateralis were performed under local anesthesia by a specialized surgeon in nine participants. A biopsy of approximately 140 mg of muscle was collected at one-third of the distance from the upper margin of patella to the anterior superior iliac spine with Weil-Blakesley forceps. A part of the sample containing well-identified fascicles was well oriented, included in an embedding medium (Cryomount, Histolab, Göteborg, Sweden), frozen in liquid nitrogen-precooled isopentane, and stored either in an ultra-low temperature freezer or in a liquid nitrogen tank. Serial 10-µm thick transverse sections were cut at -18°C using a cryostat (CM1950, Leica biosystems, Wetzlar, Germany), mounted on five serial glass slides, air-dried at room temperature, and stored at -20°C.

Immunohistochemical stains

As displayed in Tab.1, five slides were stained with 5 different cocktails of antibodies, respectively referred to as method 1 (M1), method 2 (M2), method 3 (M3), method 4 (M4), and method 5 (M5). M1 and M3 aimed to identify on a single muscle section: capillaries, complete fiber type distribution, and laminin. M1 consisted of a two-step procedure using a mouse monoclonal CD31 antibody (clone JC70A, IgG1 isotype, Dako, Agilent Technologies, Santa Clara, USA) whereas M3 consisted of a single-step procedure using a rabbit polyclonal CD31 antibody (Ab28364, IgG isotype, Abcam, Cambridge, UK) (see Tab.2). The two-step M1 procedure consisted of CD31 application in a first step, followed by the application of a cocktail of the other antibodies in a second separate step (Tab.2). The single-step M3 procedure consisted of the application of CD31 in the same cocktail as the other antibodies. M2 and M4 aimed to identify capillaries and laminin (without fiber type staining), both using a single-step procedure. M2 used the mouse monoclonal CD31 antibody (Clone JC70A, Dako) whereas M4 used the rabbit polyclonal CD31 antibody (Ab28364, Abcam) for identification of capillaries. M5 only aimed to identify fiber type distribution, using a single-step procedure adapted from Bloemberg and Quadrilatero's protocols [START_REF] Bloemberg | Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis[END_REF]. All the staining procedures were verified using negative controls to ensure appropriate staining specificity. For all details about staining procedures of the different methods, see Tab.1 and Tab.2.

Tab.1: Information on primary and secondary antibodies used in the different methods

Design

Image acquisition and analysis

Muscle sections were observed under optical microscopy (DM2000, Leica microsystems, Wetzlar, Germany) with a ×20 objective, connected to a 5 Megapixels camera with active cooling (DFC450 C, Leica microsystems, Wetzlar, Germany). Leica software (LAS version 4.3.0, Leica microsystems, Wetzlar, Germany) was used for image acquisition. The microscope was equipped with a fluorescence illumination system (Lumen 200, Prior Scientific, Cambridge, UK), and with blue (Excitation: bandpass filter (BP): 360/40 nm, Emission: BP 470/40 nm), green (Excitation: BP 480/40 nm, Emission: BP 527/30 nm), orange-red (Excitation: 545/30 nm, Emission: BP 610/75 nm), and deep red (Excitation: BP: 620/60 nm, Emission: BP 700/75 nm) filter cubes. ImageJ software (NIH, Bethesda, USA) was used to merge channels and to perform image analysis. Nine regions on each muscle section were captured in order to be representative of a large part of the muscle section. Thus, a significant number of muscle fibers could be identified on each muscle biopsy. Owing to serial cross-sectioning, the exact same muscle fibers were analyzed between the 5 different methods, for each muscle sample (see Fig. 1).

The number of capillary contacts per fiber (as previously defined as "CC" [START_REF] Hepple | Quantitating the capillary supply and the response to resistance training in older men[END_REF][START_REF] Harris | The influence of endurance and resistance exercise on muscle capillarization in the elderly: a review[END_REF])) was assessed for M1, M2, M3, and M4given that M5 was only used for fiber typing but not for direct capillaries identification. For further details on CC measurements, see the review of Harris that reported the different capillary indexes used in literature [START_REF] Harris | The influence of endurance and resistance exercise on muscle capillarization in the elderly: a review[END_REF]. CC analysis was performed on 50 type I, 50 type IIA and 50 type IIX fibers -except for two muscle biopsies for which only 34 and 35 type IIX fibers were analyzed due to their low proportion. Analyses of CC were performed on single merged images for M1 and M3. For M2 and M4 that only stained capillaries and basal lamina, CC analyses required the use of a serial cross-section to identify muscle fiber type of the cells in contact with the capillaries, therefore we used M5 staining in this case to assess muscle fiber type. The same experimenter repeated twice all measurements of each image to assess intra-experimenter reproducibility of capillaries identification. The two measurements were spaced in time to avoid any memory bias of the experimenter.

Statistics

Statistical analyses and graphics were carried out using Stata software, Version 15 (StataCorp, College Station, USA) and GraphPad software (version 5.0, Prism, San Diego, USA). Continuous data were expressed as mean ± standard deviation (SD). The assumption of normality was assessed using the Shapiro-Wilk test. To measure the inter-methods and intra-experimenter agreement, intra-class correlation coefficients (ICC) and Lin's concordance correlation coefficients (CCC) were estimated, and were interpreted according to usual rules of thumbs [START_REF] Altman | Statistics in medical journals: developments in the 1980s[END_REF]): <0.2 (negligible), 0.2-0.4 (low/weak consistency), 0.4-0.6 (moderate agreement), 0.6-0.8 (substantial/good agreement), and >0.8 (excellent agreement). The results were also presented using Bland and Altman's plots (Fig. 3).

Results and discussion

We aimed to propose new reliable time-and tissue-sparing immunohistochemical staining protocols to accurately identify capillaries, muscle fiber types (I, IIA, IIX), and basal lamina, on a single muscle section. Based on works from the last decade [START_REF] Bloemberg | Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis[END_REF][START_REF] Snijders | Muscle fibre capillarization is a critical factor in muscle fibre hypertrophy during resistance exercise training in older men[END_REF][START_REF] Snijders | Muscle fiber capillarization as determining factor on indices of insulin sensitivity in humans[END_REF][START_REF] Nederveen | Skeletal muscle satellite cells are located at a closer proximity to capillaries in healthy young compared with older men[END_REF][START_REF] Nederveen | The influence of capillarization on satellite cell pool expansion and activation following exercise-induced muscle damage in healthy young men[END_REF][START_REF] Tan | Skeletal muscle fiber-type-specific changes in markers of capillary and mitochondrial content after low-volume interval training in overweight women[END_REF][START_REF] Moro | Low skeletal muscle capillarization limits muscle adaptation to resistance exercise training in older adults[END_REF], we proposed here two new staining methods: M1 and M3 (as detailed in Tab. 1 and Tab.2).

First, the concordance between JC70A and Ab28364 antibodies both directed against PECAM-1 had to be estimated. JC70A is a mouse monoclonal antibody and Ab28364 is a rabbit polyclonal antibody, developed by different companies and with different isotypes (see Tab.1 and Material and methods). Thus, differences in capillaries identification could have been expected. From a visual point of view, Fig. 1 shows slightly sharper staining with JC70A monoclonal antibody compared to Ab28364 polyclonal antibody. This observation led us to question a potential overestimation of capillaries using the polyclonal antibody, even if the experimenters who are familiar with capillary analyses should probably not confound capillaries with artefacts. An analysis of ICC was performed to assess concordance between CC measurements from these different antibodies. Observation of individual values may also be of clinical interest. As displayed in Fig. 2, a strong similarity in CC measurements was observed for each muscle sample using the different methods. This reinforces again the strong similarity in capillaries identification using JC70A or Ab28364 antibodies, even at an individual level. Concordance between single and serial cross-section capillary identification also had to be assessed.

Tab.3: Capillary supply assessments between the different methods

Differences could have been expected between methods that require a single section analysis (M1 and M3) compared to methods that required a serial cross-section analysis (M2 and M4). As observed in Fig. 1, capillaries seem to be less apparent in M1 and M3 because of the colors of surrounding fibers, compared to M2 and M4. Indeed, capillaries seem to be very easy to detect in M2 and M4 for which fiber distribution was observed in a separate slide (M5). This first visual observation tends to suggest that capillarization might be underestimated in M1 and M3. However, capillaries should still be well detected when carefully analyzing large-format images on a large screen, despite colors of the surrounding fibers. Indeed, results showed a high degree of concordance between methods for fibers of type I (ICC: 0.89), type IIA (ICC: 0.93), type IIX (ICC: 0.88), and for all fiber types (ICC: 0.95). In addition, high reliability was also observed in CC values of each individually observed sample (Fig. 2), despite small variations. This small variability could be partly explained by the distance (a few tens or hundreds of micrometers) between muscle sections that were stained by the different methods. Although this is supposed to introduce a minor bias, the fact remains that capillaries can evolve slightly within a few micrometers. This small variability could also be attributed to a random error related to experimenter's reproducibility in the detection of capillaries, which led us to question intra-experimenter reproducibility. Lin's CCC were calculated between two separate analysis (A1 and A2) of the same images and performed by the same experimenter, in order to test intra-experimenter reproducibility. As displayed in Tab.4, Lin's CCC were systematically higher than 0.96 for each type of fibers and each method.

Measurements of capillary contacts for each sample between methods in type I

Considering all types of fibers, Lin's CCC [CCC95%] of 0.996 [0.990;1.002] was observed for M1, 0.989 [0.974;1.004] for M2, 0.987 [0.970;1.005] for M3, 0.994 [0.986;1.002] for M4, and 0.992 [0.987;0.997] for all methods, thus demonstrating a high degree of intra-experimenter reproducibility. Bland and Altman's graphical analyses were also performed to assess reproducibility between the first analysis (A1) and the repeated analysis (A2) (Fig. 3). Differences between A1 and A2 were plotted against means between A1 and A2, for type I, IIA, IIX fibers, and for all types of fibers (Fig. 3), in order to assess global agreement between A1 and A2. First, Fig. 3 shows that data were homogeneously dispersed. Then, the average agreement was very close to zero for all fiber types, meaning that A1 and A2 measurements give the same results, without systematic error. Bland and Altman's method also defines the 95% limits of agreement to help in the analysis of random error. Based on CC values already existing in the literature [START_REF] Proctor | Oxidative capacity of human muscle fiber types: effects of age and training status[END_REF][START_REF] Gavin | Lower capillary density but no difference in VEGF expression in obese vs. lean young skeletal muscle in humans[END_REF][START_REF] Kryger | Resistance training in the oldest old: consequences for muscle strength, fiber types, fiber size, and MHC isoforms[END_REF][START_REF] Groen | Skeletal muscle capillary density and microvascular function are compromised with aging and type 2 diabetes[END_REF][START_REF] Verdijk | Resistance Training Increases Skeletal Muscle Capillarization in Healthy Older Men[END_REF][START_REF] Galusca | Reduced fibre size, capillary supply and mitochondrial activity in constitutional thinness' skeletal muscle[END_REF][START_REF] Moro | Low skeletal muscle capillarization limits muscle adaptation to resistance exercise training in older adults[END_REF], we had a priori defined acceptable intervals of 0.4 capillary per fiber. Fig. 3 shows the limits of agreement between 0.1 to 0.2 capillary per fiber. Based on clinical and biological considerations, limits of agreement were therefore acceptable, since 95% of the differences between one measurement to the other were included in a range < 0.2 capillary per fiber. Finally, both Lin's CCC and Bland and Altman's graphics showed a high degree of intra-experimenter reproducibility. Ab28364 rabbit polyclonal antibody was a slightly lower sharpness compared with M1, likely due to antibody polyclonality. Therefore, we recommend to an untrained experimenter to use M1 (CD 31 JC70A) for easier detection of capillaries. Yet, high values of ICC were obtained between the different methods, and CD31 Ab28364 was already used in the literature [START_REF] Snijders | Muscle fibre capillarization is a critical factor in muscle fibre hypertrophy during resistance exercise training in older men[END_REF][START_REF] Snijders | Muscle fiber capillarization as determining factor on indices of insulin sensitivity in humans[END_REF][START_REF] Nederveen | Skeletal muscle satellite cells are located at a closer proximity to capillaries in healthy young compared with older men[END_REF][START_REF] Nederveen | The influence of capillarization on satellite cell pool expansion and activation following exercise-induced muscle damage in healthy young men[END_REF][START_REF] Tan | Skeletal muscle fiber-type-specific changes in markers of capillary and mitochondrial content after low-volume interval training in overweight women[END_REF]). On the other hand, the single-step M3 procedure was also found reliable and might be relevant for fast staining by trained experimenters. Another limitation might be the small individual variability observed in Fig. 2. Nevertheless, this no longer exists by calculating the means of several individuals, which is usually done in clinical studies (Tab.3). Based on the present results, these limitations should be tempered considering the high reliability observed between the different methods.

Tab.4: Intra-experimenter reproducibility for the different methods

Capillary contacts (CC)

Types of fibers

M1

Our initial objective was to develop a method allowing easy identification of capillaries with simultaneous staining of the three types of fibers and basal lamina. The statistical analyses were therefore conducted on the CC local indexa fiber-type dependent index, and results showed strong reliability in the identification of capillaries. Thus, the two immunohistochemical techniques M1 and M3 can be used for capillaries counting of both global indexes (CD, C/F) and local indexes (CC, CAFA, CFPE). While reliability has been verified for such indexes that only implies capillaries identification, it remains to be confirmed for indexes related to the geometry of capillariessuch as LC/PF or CapTor.

We would prefer the M1 procedure to perform morphological analyses of capillaries, since these kinds of analyses require a high level of accuracy. Indeed, from our visual observations, the M1 procedure allows providing sharp images with high quality and almost no artefact.

In perspective, it seems also important to consider the recent literature reporting an increasing number of semi-or fully automatic computational programs that are designed to perform faster and more objective analyses. Most of the time, these kinds of programs allow analyses of muscle fibers distribution and/or cross-sectional areas of fibers [START_REF] Bergmeister | Automated muscle fiber type population analysis with ImageJ of whole rat muscles using rapid myosin heavy chain immunohistochemistry[END_REF][START_REF] Wen | MyoVision: software for automated high-content analysis of skeletal muscle immunohistochemistry[END_REF][START_REF] Kastenschmidt | QuantiMus: A Machine Learning-Based Approach for High Precision Analysis of Skeletal Muscle Morphology[END_REF][START_REF] Desgeorges | Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle[END_REF][START_REF] Encarnacion-Rivera | Myosoft: An automated muscle histology analysis tool using machine learning algorithm utilizing FIJI/ImageJ software[END_REF]), but some free opensource programs (ImageJ, Fiji, CellProfiler…) are even able to perform some capillary analyses [START_REF] Smith | SMASHsemi-automatic muscle analysis using segmentation of histology: a MATLAB application[END_REF][START_REF] Mayeuf-Louchart | MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool[END_REF][START_REF] Kissane | Integrated method for quantitative morphometry and oxygen transport modeling in striated muscle[END_REF][START_REF] Sanz | Muscle2View, a CellProfiler pipeline for detection of the capillary-to-muscle fiber interface and high-content quantification of fiber type-specific histology[END_REF]). In addition to the staining methods described in the present article, it could be very useful to consider the use of such automated programs to further reduce the time for experimentation and analysis. However,

Fig. 1

 1 Fig.1 Immunohistochemical staining of human skeletal muscle

Fig. 2

 2 Fig.2 Individual values between methods by type of fibers

Fig. 3

 3 Fig.3 Graphics of Bland and Altman of intra-experimenter reproducibility in capillary contacts

two-step procedure (CD31 application followed by the cocktail of the other antibodies in a second step) Time M2, M3, M4, and M5: single-step procedure (CD31 directly applied in the cocktail of the other antibodies) Time

			Primary antibody									Secondary antibody
	Protocol	Metho d	Identification	Antibody	Reference	Specie s	Clonality	Isotyp e	Dilutio n	Source	Referenc e	Specie s	Isotyp e	Source	Dilution
	Two-step Air dry sections at room temperature M1 Capillaries Type I fibers Fixation in acetone bath Sections encircling with hydrophobic pen Anti-CD31 40 min Anti-CD31 -JC70A Air dry sections at room temperature Mouse Mono IgG1 Anti-MHCI 15 min Fixation in acetone bath BA-F8 -AB_1057225 3 Mouse Mono IgG2b 10 min Sections encircling with hydrophobic 1:40 1:100 40 min Dako 15 min DSHB 10 min pen Type IIA fibers Anti-MHC (all but IIX) BF-35 -AB_2274680 Mouse Mono IgG1 1:100 DSHB PBS wash 3×5 min PBS wash 3×5 min Blocking with PBS and GS 10% + BSA 20 min Blocking with PBS and GS 10% + BSA 20 min	546 -A-21123 350 -A-21140 488 -A-21121	GaM GaM GaM	IgG1 IgG2b IgG1	ThermoFisher Alexa Fluor ®	1:300
	Laminin Apply primary antibody: CD31 JC70A Anti-laminin 10%	2E8 -AB_2134060 10% 60 min Apply first antibody cocktail: BA-F8 Mouse Mono IgG2a 1:100 60 min DSHB	633 -A-21136	GaM	IgG2a
	Single-step Single-step PBS wash M2 M3 Blocking with PBS and GS 10% + BSA Capillaries Anti-CD31 Laminin Anti-laminin Capillaries Anti-CD31 3×5 min Anti-CD31 -JC70A and/or BF-35 and/or 2E8 and/or CD31 Mouse Mono IgG1 1:40 2E8 -AB_2134060 Mouse Mono IgG2a 1:100 Ab28364 and/or CD31 JC70A (See Tab.1) Anti-CD31 -Ab28364 Rabbit Poly IgG 1:30 PBS wash 4×5 min Dako DSHB Abcam 20min Blocking with PBS and GS 10% + BSA 30 min Type I fibers Anti-MHCI BA-F8 -AB_1057225 3 Mouse Mono IgG2b 1:100 DSHB 10% 10% Apply appropriate secondary antibody: 60 min Apply appropriate secondary antibody 60 min 546 IgG1 (dark) cocktail: 350 IgG2b and/or 488 IgG1 (dark) Type IIA fibers Anti-MHC (all but IIX) BF-35 -AB_2274680 Mouse Mono IgG1 1:100 DSHB and/or 546 IgG1 and/or 546 IgG and/or Laminin Anti-laminin 2E8 -AB_2134060 Mouse Mono IgG2a 1:100 DSHB 633 IgG2a (See Tab.1) Single-step M4 Capillaries Anti-CD31 Anti-CD31 -Ab28364 Rabbit Poly IgG 1:30 Abcam PBS wash 4×5 min PBS wash 3×5 min Blocking with PBS and GS 10% + BSA 30 min Mounting with ProLong ™ Gold Laminin Anti-laminin 2E8 -AB_2134060 Mouse Mono IgG2a 1:100 DSHB 10% Antifade Apply primary antibody cocktail: BA-F8 + 60 min	546 -A-21123 633 -A-21136 546 -A11035 350 -A-21140 488 -A-21121 633 -A-21136 546 -A11035 633 -A-21136	GaM GaM GaR GaM GaM GaM GaR GaM	IgG1 IgG2a IgG IgG2b IgG1 IgG2a IgG IgG2a	ThermoFisher Alexa Fluor ® ThermoFisher Alexa Fluor ® ThermoFisher Alexa Fluor ®	1:300 1:300 1:300
	Single-step BF-35 + 2E8 M5 PBS wash Blocking with PBS and GS 10% + BSA Type I fibers Anti-MHCI (dark) 4×5 min BA-F8 -AB_1057225 3 30 min Type IIA fibers Anti-MHC (all but IIX) BF-35 -AB_2274680 10% Apply appropriate secondary antibody 60 min	Mouse Mouse	Mono Mono	IgG2b IgG1	1:100 1:100	DSHB DSHB	350 -A-21140 488 -A-21121	GaM GaM	IgG2b IgG1	ThermoFisher Alexa Fluor ®	1:300
	cocktail: 350 IgG2b + 488 IgG1 + 633	(dark)
	IgG2a
	PBS wash				3×5 min
	Mounting with ProLong ™ Gold Antifade
	(P36934	ThermoFisher	Scientific,
	Waltham, USA)
	TOTAL				8 hours 10 min				4 hours 45 min
	BSA: Bovine serum albumin, GS: Goat serum, M: Method, PBS: Phosphate buffered saline

DSHB: Developmental studies hybridoma bank, GaM: Goat anti-Mouse, GaR: Goat anti-Rabbit, MHC: Myosin heavy chain Tab.2: Protocols of the single-and two-step procedures M1:

  Tab.3 showed high values of ICC between methods for type I fibers (ICC [ICC95%]: 0.89 [0.72;0.96]), type IIA fibers (ICC [ICC95%]: 0.93 [0.81;0.97]), type IIX fibers (ICC [ICC95%]: 0.88 [0.71;0.96]), and for all fiber types (ICC [ICC95%]: 0.95 [0.86;0.98]); thus demonstrating a high degree of similarity in capillaries identification.

although these methods undoubtedly represent the future of histomorphometry, today they require, by the users' own admission, high quality and artifact-free biopsies, which is not always the case with the muscle biopsies of patients, for example [START_REF] Sanz | Muscle2View, a CellProfiler pipeline for detection of the capillary-to-muscle fiber interface and high-content quantification of fiber type-specific histology[END_REF].

In conclusion, we propose here two new reliable, time-efficient, and timesaving immunohistochemical methods (referred to as M1 and M3 in Tab.1 and Tab.2) for capillary supply analysis in human skeletal muscle. These immunohistochemical methods allow simultaneous identification of capillaries, type I, IIA, IIX fibers, and basal lamina. The main advantage of the M1 procedure is its accuracy facilitating the identification of capillaries by an unexperienced experimenter or even potentially by an automated program, and the main advantage of the M3 procedure is its ability to perform fast staining in a single step (< 5 hours). A high degree of similarity in capillaries measurements for the different methods was found, and a high degree of intra-experimenter reproducibility was observed. The major benefit of these single section methods is the suppression of the time-consuming analysis of serial sectioning analyses.

With these methods, all the necessary information for a fine fiber typing (3 different types of muscle fibers), assessment of global and local indexes of capillarization, and measurement of muscle fibers morphometry (such as cross-sectional area thanks to the staining of basal lamina) just have to be "read" on the merged picture.