In situ checklist (Conlon lab)

Protocol checklist submitted by VGP from Conlon lab protocols [1]

link to protocol checklist word document [2]

Date:__________ Species:_________ Probes:______________________________

Day 1

0.5 vol DEPC 5min RT ___ ___ ___

PBST 5min RT ___ ___ ___ ___ ___

Proteinase K 15min RT ___

*De-vitellinization 10min ___

PBST 5min RT ___ ___ ___

Triethanolamine 5min RT ___ ___(4ml)

Acetic Acid Anhydride 5min RT ___ ___

PBST 5min RT ___ ___

4% PFA 20min RT ___

PBST 5min RT ___ ___ ___ ___ ___

20%Hyb/80%PBST 5min RT ___

Hyb Buffer o/n 60 ___

Day 2

Probe(s):

Day 3

Hyb Buffer 10min 60 ___

2xSSC 20min 60 ___ ___ ___ ___

0.2xSSC 60min 60 ___ repeat if necessary ___

1xMAB 15min 60 ___

1xMAB 15min RT ___

1xMAB/2%BMBR 60min RT ___

Blocking Buffer 60min RT ___

Antibody o/n 4 ___

Day 4

1xMAB 60min RT ___ ___ ___ ___

1xMAB o/n RT ___

Day 5

AP Buffer 5min RT ___ ___

BM Purple AP Substrate 1-8hrs RT/4 ___

PBS 5min RT ___ ___ ___

Fix:_________________ 30min RT ___ ___

MeOH 60min RT ___ (stored ___)

Day 6

Bleaching _____ RT/4