20190417: IHC IgM Swine sciatic nerve teasing (26-3-19):

Protocol:

• Fixation: in acetone -20ºC  for 10 min at RT
• Wash 2 times with PBS (5 min) with agitation
• Use the marker (DakoPen) to highlight the location of the sample.
  Indicate the sample number and the date in pencil on the frosted glass  and place the samples in the humid chamber.
• Blocking: cover the entire sample with Goat Serum 5% with triton 0.1% . Incubate 1 hour at RT in a humid chamber .
• Dilute the serum samples  1/100 in Goat Serum 5% with triton 0.1% –> add diluted serum and incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with 5% with triton 0.1% –> Add the  commercial diluted antibody  and incubate  1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Secondary antibodies: dilute GAC488 and IgM GAH 594 1/1000 with 5% with triton 0.1% –> incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Mount with Fluoromount

Results: