Frozen sections of the soleus muscle (10 µm) were blocked with 5% normal goat serum in phosphate-buffered saline containing 0.1% Triton X-100 for 30 min at room temperature and incubated for 120 min at room temperature with anti-MHC I (1:300, dilution, BA-F8, Developmental Studies Hybridoma Bank, Iowa, USA) and anti-MHC IIa (1:300 dilution, SC-71, Developmental Studies Hybridoma Bank) antibodies. Sections were washed three times with phosphate-buffered saline for 5 min each and then incubated with Alexa Fluor 555-conjugated goat anti-mouse Ig2b (1:1,000 dilution, Invitrogen, Carlsbad, CA) for MHC I or Alexa Fluor 488-conjugated goat anti-mouse IgG1 (1:1,000 dilution, Invitrogen) for MHC IIa for 60 min at room temperature. Sections were washed and covered with Fluoromount/Plus (COSMO BIO CO., LTD., Tokyo, Japan). The fluorescence images were detected and captured with a FLUOVIEW FV10i laser scanning confocal microscope (OLYMPUS, Tokyo, Japan). The number of muscle fibers and cross-sectional areas were determined in 3 randomly selected square fields (840 × 840 µm) from each section using WinROOF image processing software (Mitani Corp., Tokyo, Japan).
Ba f8
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
BA-F8 is a monoclonal antibody that recognizes the CD8 molecule on the surface of T cells. It is a purified immunoglobulin G (IgG) antibody produced by a mouse hybridoma cell line. BA-F8 is commonly used in flow cytometry and other immunological applications to identify and study CD8+ T cells.
Automatically generated - may contain errors
Lab products found in correlation
Sc 71, by Developmental Studies Hybridoma Bank (18 mentions)
Bf f3, by Developmental Studies Hybridoma Bank (12 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Alexafluor350 anti igg2b, by Thermo Fisher Scientific (2 mentions)
Alexafluor488 anti igg1, by Thermo Fisher Scientific (2 mentions)
Alexafluor594 anti igm, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 350 anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Anti mouse igm alexa fluor 555, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 anti mouse igg1, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 igg1 for mhciia, by Thermo Fisher Scientific (2 mentions)
Ab11575, by Abcam (2 mentions)
Eclipse ti microscope, by Nikon (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Fluoview fv10i confocal laser scanning microscope, by Olympus (1 mentions)
Alexafluor 488 conjugated goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Fluoromount plus, by Cosmo Bio (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
A 21244, by Thermo Fisher Scientific (1 mentions)
26 protocols using ba f8
1
Muscle Fiber Typing and Quantification
2
Skeletal Muscle Fiber Type Characterization
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Acetone-fixed 10 μm cryosections of gastrocnemius were rinsed in phosphate-buffered saline with 1% Tween 20 (PBS-T). After washing with PBS-T, the tissue sections were blocked with blocking mouse IgG (Vector Laboratories) and 5% normal goat serum (Thermo Fisher Scientific). The sections were incubated with anti-myosin heavy chain (MHC) I (1:250), anti-MHC IIa (1:250), anti-MHC IIb (1:250) (BA-F8, SC-71, BF-F3; Developmental Studies Hybridoma Bank), and anti-laminin (1:500; Sigma Chemical) antibodies overnight at 4 °C. The following day, the sections were rinsed with PBS-T and incubated with the appropriate secondary antibody conjugated to Alexa Fluor 350, 488, 555, and 647 antibodies, respectively (A21140, A21042, A21127, A21244; Thermo Fisher Scientific). Slides were mounted with ProLong Gold Antifade Mountant (P36930; Thermo Fisher Scientific) and glass coverslips. Fluorescent images were captured using a DMi8 inverted microscope (Leica).
3
Muscle Fiber Type Analysis via Immunofluorescence
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The frozen excised soleus and muscle tissues were cut centrally along the width and embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Continuous frozen sections sliced to a thickness of 10 μm were prepared using a cryostat (HM 550; Thermo Fisher Scientific, Rockford, IL, USA). Double-color immunofluorescent staining was performed to evaluate the diameter of each type 1 and 2b fiber as described previously [18] . The muscle sections were incubated with the following primary antibodies: antitype I myosin heavy chain (MyHC) isoform IgG2b (MyHCI; 1:200; BA-F8; Developmental Studies Hybridoma Bank [DSHB], The University of Iowa, Iowa City, IA, USA), antitype IIb MyHC isoform immunoglobulin (Ig)G1 (MyHCIIb; 1:100; F18; DSHB), and antilaminin IgG2a (1:200; 2E8; DSHB) at 4°C overnight. After washing, the sections were incubated with the following fluorescent-labeled secondary antibodies at room temperature for 1 h: Alexa Fluor 488-labeled goat anti-mouse IgG2b (1:500; Invitrogen Corporation, Carlsbad, CA, USA), DyLight 405-labeled goat anti-mouse IgG1 (1:500; Invitrogen Corporation), and Alexa Fluor 488-labeled goat anti-mouse IgG2a (1:500; Invitrogen Corporation). For quantification, the cross-sectional areas of the specimens were evaluated using 300 fibers in 2 visual fields at ×100 magnification per animal by using an image analyzer (WinRoof).
4
Muscle Fiber Typing Immunofluorescence
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Fiber typing followed the methods described in (69 (link)). Frozen muscle sections (10μm) of the gastrocnemius muscle were fixed in ice cold acetone for 5 minutes, rinsed in PBS, and then blocked in 1% bovine serum albumin and 10% fetal bovine serum in PBS for 1 hr. For fiber typing, sections were incubated with primary antibodies BA-F8 (1:10), SC-71 (1:30), and BF-F3 (1:10); all from Developmental Studies Hybridoma Bank, Iowa City, IA) overnight at 4°C. Sections were rinsed in PBS plus 1% bovine serum albumin and then incubated with secondary antibodies AlexaFluor350 anti-IgG2b, AlexaFluor488 anti-IgG1, and AlexaFluor594 anti-IgM (A21140, A21121, and 1010111, respectively; Life Technologies) (all used at 1:500) for 1 hr. Following secondary incubation, sections were again rinsed in PBS plus 1% bovine serum albumin. All primary and secondary antibodies were diluted in a 1% bovine serum albumin PBS solution. All steps were carried out at room temperature using room temperature reagents except where noted. All samples were fixed in ProLong® Gold Antifade Mountant (P36930; Thermo Fisher Scientific, Waltham, MA) and imaged on a Keyance microscope. Type 2B, Type 2A, and Type 1 fibers were quantified and expressed as the % of total myofibers per muscle section.
5
Immunofluorescence Analysis of Skeletal Muscle
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Histological cross sections (8 μm) were collected from skeletal muscles using a cryostat and maintained in blocking solution (10% goat serum diluted in PBS) for 30 minutes in a humid chamber. Slides were stained overnight at 4°C with PBS containing either or a combination of anti-Myh7 (BA-F8, 1:50, Developmental Studies Hybridoma Bank [DSHB]) primary antibody, anti-Myh2 primary antibody (SC-71, 1:20, DSHB), anti-Myh4 primary antibody (BF-F3, 1:10, DSHB) and with anti-laminin antibody (1:200, L9393, MilliporeSigma) to delineate myofiber outlines present in a muscle section. Immunoglobulin M (IgM) primary antibody conjugated to FITC (1:300, SAB4700348, MilliporeSigma) was used to identify myofibers with compromised membrane integrity. Primary antibodies were visualized using Alexa Fluor 568 goat anti-mouse IgG2b (Invitrogen), Alexa Fluor 488 goat anti-mouse IgG2b (Invitrogen), Alexa Fluor 674 anti-mouse IgG1, Alexa Fluor 568 IgGm, and Alexa Fluor 405 or 568 goat anti-rabbit IgG secondary antibodies diluted 1:500 in PBS. Immunofluorescence images were captured using a Nikon Eclipse Ti microscope. Entire muscle sections were analyzed for type I positive fibers. All experiments were performed in a blinded fashion whereby the experimenter was only made aware of the genotypes after the quantification was performed.
6
Muscle Protein Expression Analysis
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Primary antibodies against SERCA2a (2A7-A1), PLN (2D12), dynamin 2 (PA5-19800) and RyR (MA3-925) were obtained from Pierce Antibodies. The primary antibody for SERCA1a (A52) was a kind gift from Dr David MacLennan (University of Toronto) (Zubrzycka-Gaarn et al., 1984 (link)). The primary antibody directed against SLN was generated by Lampire Biological Laboratories (Fajardo et al., 2013 (link)). Anti-ubiquitin (P4D1) and anti-nitrotyrosine (189542) antibodies were obtained from Cell Signaling Technology and Cayman Chemicals, respectively. The primary antibody against α-actin (A4700) was obtained from Sigma-Aldrich. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), MHCIIb (BF-F3), embryonic MHC (BF-F6) (Schiaffino et al., 1989 (link); Lucas et al., 2000 (link)) and dystrophin (3B7) (Nguyen thi et al., 1990 (link)) were obtained from Developmental Studies Hybridoma Bank. Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG2b, Alexa Fluor 488 anti-mouse IgG1 and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes.
7
Fiber Type Composition Analysis
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Whole soleus was removed and a portion of the entire circumference around the mid‐belly was used. Muscles were embedded in O.C.T. compound (Tissue‐Tek), frozen in liquid nitrogen‐cooled hexane, stored at −80°C, and cut into 10 μm thick cryosections with a cryostat (Leica CM3050S, Germany) maintained at −20°C. Immunofluorescence analysis of myosin heavy chain (MHC) isoform expression was performed with primary antibodies against MHCI (BA‐F8) and MHCIIa (SC‐71), both of which were purchased from the Developmental Studies Hybridoma Bank (University of Iowa). Secondary antibodies (Alexa Fluor 350 IgG2b for MCH1 and Alexa Fluor 555 IgG1 for MHCIIa) and Alexa Fluor 488‐conjugated wheat germ agglutinin were purchased from Invitrogen. Muscle fibers negative for MHCI and MHCIIa were recognized as combination of type IIb and IIx fibers. Percentages of fiber types were calculated by counting muscle fibers positive or negative for each MHC isoforms.
8
Immunofluorescent Fiber Typing of Mouse Soleus
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Immunofluorescent fiber staining of mouse soleus muscles was adapted from Bloemberg and Quadrilatero (2012) . Soleus muscles of mice were excised and frozen in OCT in an isopentane slurry over liquid nitrogen and stored at -80 C. The muscles were sectioned on a cryostat at 10 mM thickness and mounted onto slides. Sections were blocked in 10% horse serum in PBS for 1 hour. Primary antibody mixtures containing MHCI (BA-F8, 1:50), MHCIIa (SC-71, 1:600) and MHCIIb (BF-F3, 1:100) from the Developmental Studies Hybridoma Bank (University of Iowa) were diluted in 10% horse serum and applied to the sections for 2 hours. Sections were washed 3 x 5 minutes in PBS before being incubated for 1 hour with a second antibody cocktail containing Alexa Fluor 488 IgG2b (Life Technologies, green, 1:500), Alexa Fluor 555 IgG1 (Life Technologies, red, 1:500), Alexa Fluor 350 IgM (Life Technologies, blue, 1:500) and Lectin from Triticus vulgaris-Atto 488 conjugate (Sigma, green, 1:500). Sections were washed 3 x 5 minutes in PBS and mounted using Immuno-Fluore mounting medium (MP Biomedical). Slides were imaged using the Zeiss Axio Vert A1 microscope across the entire cross-section and assembled into a composite image using ImageJ. Fibers were manually counted using the lectin stain to denote cell boundaries and assigned as being green (type 1), red (type 2a), blue (type 2b) or unstained (type 2x).
9
Quantifying Muscle Fiber Isoforms
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Isoform expression of myosin heavy chain (MHC) in the soleus was determined by an immunofluorescence as described previously [22 (link)]. Primary antibodies against MHC I (BA-F8) and MHC IIa (SC-71) were purchased from the Developmental Studies Hybridoma Bank (University of Iowa). Secondary antibodies (Alexa Fluor 350 IgG2b for MHC I and Alexa Fluor 555 IgG1 for MHC IIa) were purchased from Invitrogen. Wheat germ agglutinin conjugated with Alexa Fluor 488 (Invitrogen) was used to stain plasma membrane of each muscle fibre. A combination of type IIb and IIx fibres was recognized as muscle fibres negative for MHC I and MHC IIa. Muscle fibres positive or negative for each MHC isoform were counted and presented as percentage of fibre types.
10
Immunostaining and Muscle Fiber Size Analysis
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The mouse monoclonal antibodies specific for eMyHC (BF45) and type 1 MyHC (BAF8) were purchased from Developmental Studies Hybridoma Bank. Tissues were cryo-sectioned at 10 μm and immunostained using antibodies specific for eMyHC (1:10) or type 1 MyHC (1:5) overnight at 4 °C and processed as before69 (link). For fiber size measurements, the tissue sections were stained with WGA, (fluorophore conjugated, 1:100, Cat.# W11262, Life Technologies). Images were obtained using a Zeiss LSM 510 on Zeiss Axiovert 100 M Base and processed using NIS Elements. The minimal “Feret’s” diameter variance coefficients of the muscle fiber size was calculated on the WGA stained sections using the ImageJ 1.43u program.
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