1. incubate in three washes of xylene for 3 minutes each
2. incubate in two washes of 100% ethanol for 2 minutes each
3. incubate in one wash of 95% ethanol for 2 minutes
4. incubate in one wash of 70% ethanol for 2 minutes
6. Wash twice in dH2O for 2 minutes each
7. Rinse
Antigen unmasking:
1. heat sections in unmasking solution (1mm EDTA pH 8.0) at full power (microwave) for 2 minutes to boil, then heat at low power (PL-1) for 15 minutes.
Staining
*** premake PBST stock!
1. Wash in dH2O twice for 5 minutes each, add directly onto hot solution.
2. Using blocker marker, draw boundary around tissue section on slides. Incubate in 3% Hydrogen Peroxide for 10 minutes. (denatures endogenous enzymes)
3. Wash in dH2O twice for 5 minutes each
4. Washing in PBST (PBS+0.1% Tween 20) for 5 minutes
5. Block each section with 100-400microliter blocking buffer for one hour at RT (blocking buffer: PBST+5% rabbit normal serum).
6. Remove blocking solution, apply diluted primary antibody to slides, incubate at least 1 hour at room temperature (MSR1 Ab: cat #MAB1797 from R&D, dilute1:150 in blocking buffer).
7. Remove antibody solution and wash in (1/200) PBST three times for 5 minutes each.
8. Apply 100-400microliter diluted biotinylated secondary antibody to each section (dilute 1:200 in blocking buffer), incubate at RT for 1 hour.
9. Wash slides in PBST three times for 5 minutes each.
10. Apply 100-400microliter ABC reagent to each section, incubate at RT for 30 minutes.
11. Wash slides in PBST three times for 5 minutes each.
12. Add 100-400 microliter DAB to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH2O.
14. counterstain in hematoxylin (10 seconds)
15. Rinse in dH2O until water is clear
16. Dehydrate: 10 sec in 95% ethanol --> 10 sec in 95% ethanol --> 10 sec in 100% ethanol --> 10 sec in 100% ethanol --> 10 sec in xylene --> 10 sec in xylene
17. Mount with Cytoseal Xylene Mounting medium (From VMR, cat number 48212-196).
