Immunofluorescence of Sections

1) Place frozen sections, 5 to 10µm thick, on silanated or poly-lysine coated slides

2) Wash 3 times 5 minutes in PBS, remove excess PBS with Vacuum
To check cross linking:
Wash with 9M Urea 50mM Tris pH 6.8 or
6M Guanidine-HCl pH 5.8 for 20 minutes at room temperature

3) I° antibody (1:200 of ascites) in PBS + 1% BSA for 90 minutes RT or 4° overnight

4) Vacuum away excess antibody then wash 3 times 5 minutes in PBS by putting drop of PBS onto sections

5) II° antibody (1:30 of Rhodamine conjugated) in PBS + 1% BSA for 90 minutes RT or 4° overnight

6) Vacuum away excess antibody then wash 3 times 5 minutes in PBS by putting drop of PBS onto sections

7) Fix in freshly made solution of 4% paraformaldehyde in PBS (heat until boiling to dissolve paraformaldehyde) for 20 minutes at room temperature

8) Vacuum away excess fixative then wash 3 times 5 minutes in PBS by putting drop of PBS onto sections

9) Incubate in 0.1M glycine in PBS for 30 to 60 minutes

10) Vacuum away excess glycine then wash 3 times 5 minutes in PBS by putting drop of PBS onto sections