Protocol
for X-Gal Staining of Lung Tissue
Fixation solution
100ml of 10% Formalin
200ul of gluteraldehyde
100ul NP-40
400ml PBS
X-gal staining solution
1.050g Ferrocyanide
0.825g Ferricyanide
0.050g NaDeoxycholate
0.2g MgCl2
500ul Nonidet-P40
0.5g X-gal in 5ml N,N-Dimethylformamide
495ml PBS
Post x-gal fixative solution
1.16 g NaH2PO4
0.27g NaOH
88ml H2O
10ml 40% formaldehyde
2ml 50% gluteraldehyde
pH to 7.2
Procedure
- Inflate lung with fixation buffer and then place
tissue in 10ml of fixation buffer in containers (one
for each tissue) for 2 hours at 4oC
- Rinse 5 minutes with PBS
- Slice tissue several times with scissors or cryostat
blade to increase surface area. Exercise caution to
not actually cut the tissue in half!
- Rinse containers twice with distilled water
- Fill containers with 10ml of x-gal staining solution
- Place tissues in staining solution
- Incubate for 3 hours aat 37oC and then
for 24 hours at room temperature. Alternatively, incubation
can be for 24 to 48 hours at room temperature
- Remove and wash tissues twice in distilled water
and place in post x-gal fixative solution
Note: Rat epididimas can be used as a positve control
and will turn dark blue in color. Positive samples may
not be visually positive, but will be identified as
positive from prepared slides.
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