Cryo sections:

Warm up on slide warmer 10-15’

For 1 slide (wt and ko) – 1 x40’ prewarmed 2N HCl at 37 degrees (conc is 12.1N)

Wash sodium borate 0.1M 1 x 10’

For HCl treated and amplified, block 10% GS, 0.4% Tx-100, in PBS 60’

For 1 slide, do high Tx – 10% GS, 1% Tx-100, in PBS 60’

Antibodies 2hr RT:

For HCl and amplified, antibody 1:50 in block

For high Tx, antibody 1:50 in PBS with GS10%, no Tx

Wash 3x PBS

Secondaries

HCl – anti- rabbit Cy3 1:500 in block

High Tx – anti-rabbit Cy3 1:500 in PBS with GS, no Tx

Amplified – 1 hour biotin-anti rabbit in block

Then wash 3X

Then 1 hour Cy3-SA in block

Wash 3X PBS (middle wash with Hoeschst)

Mount and view