Immunofluoresent staining of frozen sections
Fixation of lungs for sections:
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4% paraformaldehyde in PBS for 6-24h at 4C
30% sucrose in PBS for 24h at 4C
1h in OCT
Freezing of lungs in OCT with liquid nitrogen or dry ice.
Lungs are transferred to be sliced
Section are frozen at -20C
Important notes:
1. Change slide glass jar between washes
2. The staining should take place in a humid environment – place a water
soaked paper under the slides (without it touching the slides)
Staining:
1. Fix sections in 100% Acetone for 10min at R.T with gentle shaking
2. Wash in PBS for 30-60sec
3. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking
(250μl Triton into 1L PBSx1)
4. Discard washing solution and draw borders using the PAP pen
5. Block in 3% normal goat serum in PBS for 1h at R.T (300μl normal goat
serum into 10ml PBSx1)
6. Discard blocking solution.
7. Add primary antibody diluted in 3% normal goat serum in PBS (for Relm-α
1:100 dilution from 100μg/ml stock)
8. Incubate O.N at 4C
9. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking
10. Add secondary antibody diluted in 3% normal goat serum in PBS (for antirabbit IgG 1:400 dilution from 1.5mg/ml stock)
11. Incubate 1h at R.T
12. Wash in 0.025% Triton X-100 in PBS 3x 5min with gentle shaking
13. Dry slides as much as possible including removing the PAP pen borders
14. Add 10μl ready to use DAPI + mounting and cover the slide
15. Slides can be kept at 4C for a few months