in situ Protocol Day 1
Remember:
- use plastic transfer pipettes
- Thaw out paraformaldehyde and heat oven to 60°C
Steps: Wash time

Check

when done

Rehydrate Embryos
100% Methanol 5 min ____
75% Methanol + 25% H2O 5 min ____
50% Methanol + 50% H2O 5 min ____
25% Methanol + 75% PBT 5 min ____

Note: PBT = 1X PBS +

100% PBT 5 min ____

0.1% Tween-20

100% PBT 5 min ____
100% PBT 5 min ____
2 - Proteinase K (PK) Note: Stock @ 10 mg/ml
10 min ____ 100 µl PK + 100 ml PTW
3 - 0.1 M Triethenolamine pH 7-8
5 min ____ Note: 18.5g/1L H20
Measure 5 min ____
4 - Acetic Anhydride
Do not discard after; 5 min ____ Note: 250 µl/100 ml
add 250 µl more 5 min ____
5 - PBT wash
5 min ____
5 min ____
6 - Re-fix - 4% Paraformaldehyde Note: 4 ml PBT +
20 min ____ 1 ml 20% paraformaldehyde
7 - PBT wash
5 min ____
5 min ____
5 min ____
5 min ____
5 min ____
8 - Add Hybridization Buffer
3-4 hours ____ at 60°C
9 - Replace with Probe solution
Overnight ____ at 60°C
                                                in situ Protocol Day 2
Steps: Wash time
1 - Replace probe with hybridization buffer
10 min ____ at 60°C
2 - 2X SSC
(Can do just one if omitting RNAse) 20 min ____ at 60°C
20 min ____ at 60°C
20 min ____ at 60°C
3 - 2X SSC + RNAse A + RNAse T

at 37°C

Note: RNAse A 200µl/100 ml

30 min ____

      RNAse T 1µl/100 ml

4 - 2X SSC
10 min ____ Room temp
5 - 0.2X SSC
30 min ____ at 60°C
30 min ____ at 60°C
6 - 1X MAB
10-15 min ____ Room temp
10-15 min ____ Room temp
Antibody Incubation
7 - 1X MAB + 2% BMB
1 hour ____ Room temp
8 - 1X MAB + 2% BMB + Antibody (1/2000 dilution)
100 ml: 20 mL 10% BMB Overnight ____ at 4°C
             80 ml 1X MAB
             50 µl AB
                                               in situ Protocol Day 3
Steps: Wash time
1 - 1X MAB Wash (or PBT)
45 min ____
45 min ____
45 min ____
45 min ____
or overnight at 4°C (for last step) 45 min ____
                                               in situ Protocol Day 4
Chromogenic Reaction
Steps: Wash time
1 - Wash w/ Alkaline Phosphatase Buffer (100 ml = 0.6g NaCl + 10 ml 1M Tris pH 9.5 +
5 min ____ 5 ml 1M MgCl2 + 100 µl Tween20 to 100 ml MilliQ water)
2 - Transfer to glass vials
3 - Replace buffer with BM Purple
- Cover with foil
- Shake @ room temperature (Check every 30 min for signal for 2 hours at
room temp or leave o/n @ 4°C)
4 - 1X MAB wash 5 min ____
5 - Fix in MEMFA 1 hour ____ 100 ml: 80 mL H20
                       10 ml MEMFA salts
6 - 1X MAB wash 5 min ____                        10 ml Folmaldehyde
7 - Bleach 200 ml:  6.67mL H202 (30%)
onto light box (cover with foil) 2 hours ____          10 ml Formamide
      5 ml SSC (20X)
8 - 1X MAB wash (2x)
5 min ____
5 min ____
9 - Store in Methanol (-20°C)