Wax sections were de-waxed in a series of xylene, graded xylene/ethanol mixture, graded ethanol mixture, and finally incubation in PBS. The sections were stained with HE and Masson’s trichrome. Cryosections were mounted on silane-coated slides, air dried, and blocking with 5% bovine serum albumin (BSA) + 0.2% Triton X-100 in PBS if needed. Sections were then left to incubate with primary antibodies overnight at 4 °C, or 4 h at room temperature before overnight incubation at 4 °C. The primary antibody used was rabbit anti-laminin (1:200, L9393, Sigma, St. Louis, MO, USA), rabbit anti-CD31 (1:50, PA5-16301, Thermo Fisher Scientific), rabbit anti-Phospho-ERK1/ERK2 (1:50, 44-680G, Thermo Fisher Scientific, Waltham, MA, USA), and rabbit anti-GluR1 (SD2010) (1:100, MA5-32344, Thermo Fisher Scientific). The secondary antibody for fluorescent photography was CF™ 488 goat Anti-Rabbit IgG (H+L) used (1:400, SAB4600045, Sigma). Primary antibody omission controls were used for all immunostaining protocols to control nonspecific binding. Fluorescent was performed with a Zeiss Axioscope microscope equipped with charge-coupled device (CCD) camera with appropriate filter sets.
L9393
Manufactured by Merck Group
Sourced in United States, Japan, Sweden, United Kingdom
The L9393 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of the L9393 is to provide a reliable and consistent platform for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
A11008, by Thermo Fisher Scientific (11 mentions)
Sc 71, by Developmental Studies Hybridoma Bank (6 mentions)
Bf f3, by Developmental Studies Hybridoma Bank (6 mentions)
Laminin, by Merck Group (5 mentions)
Z0334, by Agilent Technologies (5 mentions)
Ab15580, by Abcam (5 mentions)
D1306, by Thermo Fisher Scientific (5 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (4 mentions)
Ab13970, by Abcam (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Ba d5, by Developmental Studies Hybridoma Bank (3 mentions)
T6199, by Merck Group (3 mentions)
Ab6586, by Abcam (3 mentions)
A21125, by Thermo Fisher Scientific (3 mentions)
Ab6556, by Abcam (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Mab377, by Merck Group (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (3 mentions)
143 protocols using l9393
1
Immunohistochemical Characterization of Tissue Samples
2
Muscle Fiber Analysis Protocol
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TA samples were mounted in tragacanth (Sigma-Aldrich # G1128) on plastic blocks and frozen in liquid isopentane cooled in liquid nitrogen and stored at -80°C. Samples were cut into 10μm cross-sections using a cryostat at -20°C then mounted on lysine coated slides (Superfrost), as described in.69 (link),73 (link) Cross-sections were brought to room temperature, rehydrated with PBS (pH 7.2), then blocked with goat serum (10% in PBS). They were then incubated with primary polyclonal anti-laminin rabbit IgG antibody (Sigma-Aldrich # L9393, 1:500) for 1 h at room temperature. Sections were then washed three times in PBS before being incubated for 1 h at room temperature with an Alexa Fluor® 594 goat anti-rabbit IgG antibody (A-11037, 1:500). Sections were then washed three times in PBS and slides were cover slipped using Prolong™ Gold (P36930, Invitrogen) as a mounting medium. Slides were imaged with a Zeiss fluorescence microscope (Zeiss Axio Imager 2). Median distribution of minimum Feret diameters of at least 300 fibers per muscle sample were analyzed using ImageJ (NIH, Bethesda, MD).74 (link) The degree of myofiber atrophy was calculated as percent difference in mean minimum Feret diameters, relative to muscles of sham Atg7f/f mice. The means, fibers distributions, and average number of fibers analyzed per muscle per mouse are shown in Figure S6 .
3
Laminin Staining for Muscle Fiber Analysis
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Cross sections of muscles were stained with antibodies directed against laminin (1:1000, Sigma L9393;). Images were obtained using the Axio Observer Z1 from Zeiss. The minimal fiber feret is defined as closest possible distance between the two parallel tangents of an object, in our case the muscle fiber [21 (link)]. The Zeiss Zen software was used to measure the minimal fiber feret.
4
Quantifying Pax7+ Satellite Cells in mdx Mice
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Muscle samples from mdx mice where frozen in OCT. Muscle samples were sectioned at 10 microns and blocked with 5% donkey, 1% BSA, 0.25% Tween20 for 30 minutes at room temperature. A rabbit polyclonal anti-Pax7 (dilution1:500; Abcam ab34360, Cambridge, UK) was incubated overnight at 4 °C in a humidity chamber. Secondary (anti-rabbit Cy3 1:100) was incubated for 1 hour in a humidity chamber at room temperature. The tissues were blocked again with 5% Rabbit serum in 1x PBS with 1% BSA, 2hrs in a humidity chamber at room temperature. Rabbit polyclonal anti-Laminin (dilution1:500; Sigma-Aldrich L9393, St. Louis, MO) was incubated overnight at 4 °C in a humidity chamber. Secondary AB (anti-Rb 488 1:300) was incubated at room temperature for an hour in a humidity chamber. The muscle tissue was then exposed to 4% paraformaldehyde for 10 minutes at room temperature. Prolong gold with DAPI was added and the slides were cover slipped. Pax7 + satellite cells where identified by co-localization with laminin. Relative Pax7 + satellite cell numbers where quantified by utilizing ImageJ software described previously6 (link).
5
Quantification of Muscle-Resident FAPs
6
Immunostaining Assay for Neurite Outgrowth
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To explore the neurite outgrowth, the immunostaining was performed as follows. Cultured neurons were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at 4 °C and wash three times with PBS for 5 min each. To permeate the cell membrane, 1% Triton X-100 (Sigma-Aldrich) was used for 5 min at room temperature, followed by PBS washing. For blocking nonspecific binding, the sample was treated with 6% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min. After washing with PBS, it was reacted with primary antibodies diluted in 1.5% BSA solution for 2 h at 37 °C. The following primary antibodies were used: anti-MAP2 (1:500, M3696, Sigma-Aldrich), anti-tau-1 (1:500, MAB3420, Merck Millipore, MA, USA), anti-beta-III-tubulin (1:500, T2200, Sigma-Aldrich). Secondary antibodies (Alexa Fluor 488 and 594, 1:500, A11001, A11008, A11012 and A11032, Invitrogen; Thermo Fisher Scientific) diluted in 1.5% BSA were loaded for 30 min at 37 °C. To characterize damage of a biomolecule on the NIR-illuminated surface, anti-laminin (1:500, L9393, Sigma-Aldrich) was used with the same procedure except that there is no cell fixation and permeabilization step. An inverted microscope (IX71; Olympus) with a digital camera (DP71; Olympus) was used to take fluorescence and phase-contrast images.
7
Quantitative Laminin ELISA Protocol
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WGA eluates were diluted 1:50 in TBS and coated on polystyrene ELISA microplates (Costar) overnight at 4 °C. Plates were washed in LBB and blocked for 1 h in 3% BSA in LBB. Mouse EHS laminin was diluted in LBB and applied for 2 h. Wells were washed with 3% BSA in LBB and incubated for 30 min with anti-laminin antibody (L9393, Sigma-Aldrich, 1:5000 dilution) followed by HRP-conjugated anti-rabbit IgG (Invitrogen, 1:5000 dilution). Plates were developed with o-phenylenediamine dihydrochloride and H2O2, and then reactions were stopped with 2 N H2SO4. Absorbance per well was read at 490 nm by a microplate reader.
8
Immunohistochemical Analysis of Muscle Fiber Types
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The 10 μm thick transverse serial cryosections of TA muscles were cut at the mid‐belly region using Microm cryostat machine. Briefly, the tissue sections were blocked with 0.2% bovine serum albumin (BSA) and 10% normal sheep serum (NSS) in PBS‐Triton X100 (PBST) for 1 h at RT. All washes were carried out with PBS. Following washing, the sections were incubated in 5 μg/mL of mouse monoclonal primary antibody against myosin heavy chain type‐1 (A4.840s DSHB, IA, USA) in 0.2% BSA and 5% NSS antibody dilution buffer in PBST overnight at 4°C. After washing, the sections were then fixed with 10% neutral buffered formalin for 5 min at RT, washed and incubated in 1:300 dilution of biotinylated sheep anti‐mouse IgG (ab6807, Abcam, MA, USA) and 1:1000 dilution of rabbit anti‐laminin (L9393, Sigma‐Aldrich, MO, USA) in the antibody dilution buffer for 1 h at RT. Following washes, the tissue sections were then incubated in 1:400 dilution of streptavidin AF488 conjugate and goat anti‐rabbit AF594 (Invitrogen, CA, USA) and then washed and mounted with Prolong gold anti‐fade mounting medium with DAPI (Invitrogen, CA, USA). Images were obtained at ×10 and ×20 magnifications with the Olympus IX73 inverted microscope using CellSens Standard software (Olympus Soft Imaging Solution, USA) and the MyHC1 positive myofibres were quantified with ImageJ software.28
9
Visualizing Skeletal Muscle Microstructure
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Cryosections (10 μM) were obtained from mid‐section of tibialis anterior (TA) muscle from WT‐NCD, WT‐DIO and TG‐DIO mice. Laminin (sarcolemmal marker) was stained using antibody L9393 from Sigma at 3.5 μg/ml dilution to visualize sarcolemma. Capillaries were stained using CD31 at 10 μg/ml (#MCA2388 Bio‐Rad). All primary antibodies were visualized using suitable Alexa Fluor® secondary antibodies from Molecular Probes, Eugene, OR. Nuclei were stained using DRAQ5 fluorescent probe 5 μM (Thermo Scientific). Immunostained sections were examined using confocal microscopy. Myofiber number was counted using Amira software version 6.3 from FEI Hillsboro, OR. CD31 and nuclei were counted using Image J.
10
Immunostaining of Zebrafish Embryos
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Embryos for most antibodies were fixed at the indicated stage in 4% paraformaldehyde, permeabilized in PBST (PBS+0.5% Triton X-100), and blocked in PBST+2% bovine serum albumin. Antibodies and concentrations were as follows: anti-Pax2a (GTX128127, Genetex; 1:200); anti-pSmad3 (ab52903, Abcam; 1:200); anti-Laminin 1 (L9393, Sigma-Aldrich; 1:100); anti-fibronectin (F3648, Sigma-Aldrich; 1:100); anti-GFP (A10262, Invitrogen; 1:200).
In accordance with methods described by Carrara et al. (2019) (link), anti-Nidogen 1/Entactin (ab14511, Abcam) was used at 1:100 and required a modified embryo preparation after 4% paraformaldehyde fixation: embryos were permeabilized in 30 μg/ml Proteinase K for 15 min, and blocked in 0.8% PBST+10% sheep serum+1% bovine serum albumin. The antibody was applied in 0.8% PBST+1% sheep serum+1% bovine serum albumin.
Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse (A-11001, Invitrogen), Alexa Fluor 488 goat anti-rabbit (A-11008, Invitrogen), Alexa Fluor 488 goat anti-chicken (A-11039, Invitrogen), all used at 1:200. Nuclei were detected by incubation with 1 µM TO-PRO-3 iodide (T3605, Invitrogen). Embryos were cleared through a series of 30%/50%/70% glycerol (in PBS) prior to imaging.
In accordance with methods described by Carrara et al. (2019) (link), anti-Nidogen 1/Entactin (ab14511, Abcam) was used at 1:100 and required a modified embryo preparation after 4% paraformaldehyde fixation: embryos were permeabilized in 30 μg/ml Proteinase K for 15 min, and blocked in 0.8% PBST+10% sheep serum+1% bovine serum albumin. The antibody was applied in 0.8% PBST+1% sheep serum+1% bovine serum albumin.
Secondary antibodies used were: Alexa Fluor 488 goat anti-mouse (A-11001, Invitrogen), Alexa Fluor 488 goat anti-rabbit (A-11008, Invitrogen), Alexa Fluor 488 goat anti-chicken (A-11039, Invitrogen), all used at 1:200. Nuclei were detected by incubation with 1 µM TO-PRO-3 iodide (T3605, Invitrogen). Embryos were cleared through a series of 30%/50%/70% glycerol (in PBS) prior to imaging.
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