Immunohistochemistry Protocol

Description

Intended for use with frozen sections, up to 20um thick (thin is better, aim for 10um).  Works with short fix times (4%PFA for 2 hours at 4C).  An overnight fix may work for many antibodies, but we always use a short fixation time to be sure.

Method

  1. Warm slides at RT until dry.
  2. 3 X PBT (PBS + 0.1% Triton) 5min
  3. 5% HISS/PBT RT 1 hour (blocking step)
  4. Primary Ab in 1%HISS/PBT 4C overnight (as short as 2–3 hours at RT)
  5. 3 X PBT 5min
  6. Secondary Ab in 1%HISS/PBT RT 2 hours (always use 1:250 dilution)
  7. 3 X PBT 5min
  8. Brief rinse in PBS
  9. Stain nuclei with Hoechst, 1:10,000 in PBS for 10min
  10. Brief rinse in PBS
  11. Air dry
  12. mount (e.g. vectashield)

Materials

PBT - PBS + 0.1% Triton
HISS - heat inactivated sheep serum
Hoechst - 1:10,000 dilution in PBS

Troubleshooting

Note that we have used donkey serum in place of HISS above and have seen no differnence.
If having problems with background, try the following alternative Block/Hyb solution:
3%BSA/1% Donkey serum - use for block and with antibody
This has lower background than using sheep serum especially for doing neural tube.

Discussion

Hoechst Staining
We have found that DAPI staining (using Vectashield with DAPI) results in uneven staining.  This improves with time but is still a problem at the edges of the kidney.  Hoechst seems to do a better job in our hands, perhaps because it is in PBS instead of mounting media.  To use, dilute 1:10,000 in PBS and stain for 10min.  No need to add Hoechst to the plain Vectashield when mounting.  It is easiest to create a 100X stock of Hoechst in PBS and freeze aliquots for later use.