TA and gastrocnemius muscles were sectioned on a cryostat (10 μM thick) and hematoxylin and eosin-phloxine staining was performed. These sections were used to count the percentage of fibers with central nuclei using the methods previously described [1 (link)]. Additional TA muscle sections from 3 and 7 day cohorts were assessed for Mac-1 expression, a marker for leukocytes. To do this, sections were fixed in acetone, blocked using a Streptavidin/Biotin Blocking Kit (SP-2002, Vector Laboratories, Burlingame, CA), incubated in rat anti-mouse CD11b primary antibody (550282, BD Biosciences, San Jose, CA) or IgG2b (559478, BD Biosciences) for controls, followed by a biotin goat anti-rat Ig secondary antibody (559286, BD Biosciences). Antigen-antibody interactions were visualized using DAB (550880, BD Biosciences). For each slide, ten digital images were acquired at 40X magnification (Leica DM2000 and QImaging Micropublisher RTV 5.0). A single, blinded investigator assessed the % area of positive staining per image using thresholding (Image J software, National Institutes of Health, Bethesda, MD). The average % of muscles’ cross-sectional area stained positive from the 10 images per muscle section was calculated and used for statistical analyses.
3 3 diaminobenzidine (dab)
Manufactured by BD
Sourced in United States
The DAB is a laboratory equipment designed for the visualization of specific protein targets in tissue samples. It functions by converting a chromogenic substrate into a colored precipitate at the site of the target protein, enabling its detection and localization within the sample.
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5 protocols using 3 3 diaminobenzidine (dab)
1
Quantifying Muscle Fiber and Inflammatory Cell Changes
2
Immunohistochemical Staining Protocol
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Standard IHC staining was performed as previously described (20 (link)). In brief, after de-paraffinization and rehydration, sections were subjected to heat-induced antigen unmasking. Slides were then incubated with various primary antibodies at 4°C overnight, after blocking with 5% goat serum. Slides underwent color development with DAB (BD Pharmingen, New York, NJ, USA) and haematoxylin (Vector Laboratories, Burlingame, CA, USA) counterstaining. Images were captured with a microscope (BX51, Olympus, Tokyo, Japan) and processed with identical settings. Ten visual fields from different areas of each tumor were evaluated.
3
Measuring Cell Proliferation In Vivo
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To assess proliferation in vivo, mice were given 100 μg/g body weight of BrdU intraperitoneally 1.5 hours before euthanasia for tissue or tumor sampling. Paraffin sections were processed, and antigen was retrieved as detailed above. Sections were then stained with the biotinylated anti-BrdU antibody (#ab6326, clone ICR1; Abcam, Cambridge, UK) at 1/100 dilution and detected with streptavidin-horseradish peroxidase (BD Pharmingen) and DAB.
4
RV Capillary Density Quantification
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RV tissue sections were deparaffinized and stained for CD31 (550300, BD, Franklin Lakes, NJ, USA) and subsequent DAB (BD, Franklin Lakes, NJ, USA) and H&E staining. Small, non-muscularized vessels were manually counted by two independent, blinded observers using BF microscopy-images from 6 randomly selected, transversal sections. RV tissue area was calculated with ImageJ software (National Institute of Health, Bethesda, MD, USA), and counted vessels were corrected for area.
5
Immunohistochemical Analysis of Mouse Tissue
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Mouse tissue was fixed with 10% formalin overnight and kept in 70% ethanol at 4°C until processed into paraffin tissue blocks by the Imaging Core at Lerner research institute of Cleveland Clinic. Paraffin sections were subjected to heat-induced epitope retrieval, as recommended by the antibody manufacturer before staining. Deparaffinized, epitope-retrieved sections were blocked with 10% normal goat serum (Life Technologies; no. 50062Z) and then incubated with primary antibody overnight. Next day, the sections were washed with 0.05% Tween PBS followed by the incubation with secondary antibody and streptavidin-horseradish peroxidase (HRP) for immunohistochemistry or fluorescence-conjugated secondary antibody for immunofluorescence. Staining was visualized with HRP-substrate chromogen DAB (BD Pharmingen) or with confocal microscopy. The following primary antibodies were used for staining presented in the study: Ki67 (Cell Signaling Technology 12202, 1:200).
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