Differential SERCA Expression in Laryngeal Muscles

We faced unsuccessful fast fiber type's identification on frozen sections then we shifted to paraffin sections. Deparaffinized sections were incubated with 3% H2O2 in PBS for 10 minutes followed by microwave treatment (5 minutes ×3 times at 500 Watt in citrate buffer, pH 6). Then, sections were incubated in blocking solution (0.3 M glycine, 50 mM ammonium chloride, and 1% BSA in PBS) for 30 minutes before incubation in primary antibodies. Sections were incubated with anti-SERCA1 or -SERCA2 and anti-MHC I or -MHCII (Table 1) antibodies for 1-2 days at 4 ◦C, then washed, and incubated with Alexa Fluor® 488 donkey anti-mouse IgG and Fluor® 594 donkey anti-goat IgG secondary Abs for 1 hour at room temperature. Double IHC was performed using (anti-SERCA1 + anti-SERCA2) and (anti-SERCA2 + anti-MHCII) combinations to check co-expression. In double IHC, the same steps were done as before with mixture of primary or secondary antibodies with same dilutions.