Journal: Current Protocols
Article Title: Adaptable Immunofluorescence Protocol for Muscle Fiber Typing in FFPE Human and Mouse Skeletal Muscle and Intact Mouse Hindlimbs
doi: 10.1002/cpz1.70246
Figure Lengend Snippet: Effects of antigen retrieval procedures on MyHC immunolabeling. Row 1: Representative images of serial sections of fresh frozen quadriceps muscle preparations (conventional method) demonstrating immunoreactivity with antibodies to MyHC 1, MyHC 2a, MyHC 2x, and MyHC 2b. Row 2‐6: Sections of FFPE mouse muscle were stained with antibodies to MyHC 1, MyHC 2a, MyHC 2x, and MyHC 2b to evaluate the effectiveness of antigen unmasking using different antigen retrieval methods. Each image contains two muscles: plantaris on the left, soleus on the right, to include all fiber types in a single image. Row 2 shows that all MyHC antigens are masked on FFPE tissue preparations. NOTE : image brightness was enhanced for tissue visualization. Antigen retrieval methods tested on FFPE sections included HIAR using Sodium Citrate Buffer, pH 6.0 (Row 3), HIAR using EDTA, pH 8.0 (Row 4), HIAR using Tris‐EDTA, pH 9.0 (Row 5), and a combination of HIAR using EDTA, pH 8.0, followed by PIAR using Proteinase K digestion (Row 6). Immunostaining with each antibody was performed individually with antibodies against MyHC 1, MyHC 2a, MyHC 2x, and MyHC 2b. Retrieval with Tris‐EDTA, pH 9.0, and EDTA, pH 8.0, enhanced epitope accessibility for MyHC 1, MyHC 2a, and MyHC 2b, while proteinase K combined with HIAR improved staining for MyHC 2x. However, the combined HIAR and PIAR approach was not effective for labeling MyHC 1, MyHC 2a, and MyHC 2b. Overall, high‐pH antigen retrieval methods were most effective for unmasking MyHC 1, MyHC 2a, and MyHC 2b in FFPE muscle sections. Arrows indicate strongly labeled fibers. Asterisks indicate weakly labeled fibers. Scale bars = 200 µm.
Article Snippet: Monoclonal: Clone BF‐F3 , Mouse isotype IgM , MyHC 2b , 0.85 μg/ml , DSHB , AB_2266724.
Techniques: Immunolabeling, Staining, Muscles, Immunostaining, Labeling