Tzahor Lab

Immuno-Staining

1. PFA 4%  O/N
2. change to PFA 1%  
3. Paraffinization (As in the protocol- depending on embryo age)  
Deparaffinization:  
4. Deparaffinization (not age dependant):  
 
  1. Xylen
  2. EToH 100%
  3. EToH 90% (in DDW)
  4. EToH 70% (in DDW)
  5. EToH 30% (in DDW) 
  6. PBS
  7. DDW 

2X10°

2X10°

1X10°

1X10°

1X10°

3X5°

2X10°

 
Microwave treatment for Ag exposure:  
5. Put the slides in a plastic chamber and cover with 10mM Sodium citrate PH=6,
put a loose lid/tip cover, to prevent boiling over.
6. Boil until boiling starts ~2°  
7. Reduce microwave power to minimum 1X13°
  (Check every few minutes that solution hasn’t evaporated, if so add more)
8. Cool the slides in the solution 10° – 1h
9. Put in glass/plastic box. Wash with PBS 2X10°
Permibilization and 1st Ab:  
10. Permibilization
(0.25% Tryton, 1% BSA in PBS)		 1X15°
11. Blocking
(1% BSA, 5% Goat serum in PBS)		 1X1h
12 Shake from access water, dry bottom of slide and add 70ul of 1st antibody directly on the slides.
Cover gently with a cover slip.
13. Put in humid chamber at 40c  
(Slides lying in slide box and put wet paper underneath)		 O/N - 4°c


Immuno-Staining 2nd Ab (Dapi Optional)

Don’t forget: Use a proper secondary antibody according to the origin of the first!!
1. Take from 40c and put in RT (box close) 1h
2. Wash with PBS
This is done in the plastic or glass box.
After the first wash, take the slides out – gently making sure cover slips are removed!		 3X10°
3. Shake from access water, dry bottom of slide and add 70ul of 2nd antibody directly on the slides.
Cover gently with a cover slip.
(Dilution dependant on Ab, with 5% goat serum in PBS. Let stand in humidified box.
The box needs to be covered from light)
4. Put in humid chamber at RT 40°-50°RT
5. Wash with PBS
This is done in the plastic or glass box.
After the first wash, take the slides out – gently making sure cover slips are removed! 		3X10°
6. Dapi Staining (Optional):
  1. Shake from access water, dry bottom of slip and add 70ul of Dapi solution directly on slides.
    Cover with cover slips.
  2. Put in humid chamber at RT
    (1:10,000 with 1% BSA, 5% Goat serum in PBS. box covered from light)
  3. Wash with PBS
    This is done in the plastic or glass box.
    After the first wash, take the slides out, gently making sure cover slips are removed!

5°-10°  RT

3X 10°
7. Mounting with Elvanol  
8. let dry horizontaly O/N  RT

IF 2 monoclonals (from Orna Halevy):

Myoblasts were grown for 2 days and fixed in ice-cold AFA solution (70% ethanol:formaldehyde:acetic acid 20:2:1) for 1 min, then incubated with a blocking solution [1% (v/v) goat serum in PBS] for 1 h.

Cells were incubated with HV11 mouse monoclonal antibody against ventricular MHC for 2 h, followed by incubation with a biotinylated anti-mouse IgG (Fab fragment; 1:250;715-065-150 Jackson, West Grove, PA) for 1 h, and Texas-Red Streptavidin (1:300; Jackson 016-070-064) for 30 min.

EB165, a mouse monoclonal antibody against embryonic MHC was added to the cells for 2 h (IgG1, ascites fluid; 1:1000; kindly provided by E. Bandman), followed by incubation with a FITC-conjugated anti-mouse IgG antibody (1:250, Jackson 715-095-150) as the secondary antibody. Nuclei were detected by counterstaining with DAPI.

Solutions:
100mM Tri- Sodium citrate PH=6:
Mw = 294.1 g/mol
1M à 294.1g
100mM (0.1M) à 29.41g
Adjust PH to 6