Modified 5/26/09 TH


For Frozen TissueSections:


Tissue Preparation

  1. Ice cold PBS perfusion, then cut kidney in half and fix PLP for 2 hours, then 18% sucrose overnight at 4íC.
  2. Embed in OCT, and immediately put into -80íC.
  3. Cut cryosections 5-7μm thick.


X-gal stainingsolution preparation

Prepare at least one dayin advance and confirm pH of 7.4. Store at 4íC wrapped in foil. Decant thesolution before use (crystals will form). Should be used the next day for best results.


0.5M K3Fe(CN)6                   500μL

0.5M K4Fe(CN)6                   500μL

1M MgCl2                              100μL

1% Na deoxycholate                      500μL

2% NP-40                             0.5mL

50 mg/mL X-gal in DMF     1.0mL

1x PBS                                   46.9mL


Staining: Air dry slides for 30 minutes. Wash three timesin 1x PBS. Stain in X-gal solution for 6 hours to overnight (or to desiredstaining) at 37íC. You will need to establish optimal staining time. Stain inglass Coplin jar, sealed tightly with parafilm and covered with foil. Ifsolution turns green or a bluish precipitate forms, then either the solution isno good or you are using too small a volume of the solution for staining.


Wash 3 x 5 mins in 1x PBS.


Post-fix  in1% paraformaldehyde in 1x PBS for 2-5 mins.


Wash 3 x 5 mins in 1x PBS.


Then either:


Counterstain (if desired) for about 2 minutes using 0.17%Eosin Y solution in 70% EtOH. Rinse briefly in 70% EtOH, 95% EtOH x2, 100% EtOHx2, then CitriSolv x1. Mount with Permount.


OR (for immunofluorescence)


Continuewith IF protocol and mount inaqueous mounting medium (ProLong Gold). Shorter X-gal staining times decreaseIF signal quenching.