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

1. Inflate lung with fixation buffer and then place tissue in 10ml of fixation buffer in containers (one for each tissue) for 2 hours at 4^oC
2. Rinse 5 minutes with PBS
3. Slice tissue several times with scissors or cryostat blade to increase surface area. Exercise caution to not actually cut the tissue in half!
4. Rinse containers twice with distilled water
5. Fill containers with 10ml of x-gal staining solution
6. Place tissues in staining solution
7. Incubate for 3 hours aat 37^oC and then for 24 hours at room temperature. Alternatively, incubation can be for 24 to 48 hours at room temperature
8. 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.