Hi,

I am currently attempting to optimize an IHC protocol for some lymphatic endothelial cells that I'm studying. I've been having a lot of trouble getting consistently good images after staining, and am looking for any suggestions as to how I can fix this problem. I am staining for ZO-1, Claudin, Occludin, and VE-cadherin tight junction proteins.

Most of the images I get show very non-specific signal (I've attached some to this post so you can see for yourself). The proteins should look like outlines of each individual cell, but I haven't been able to see that yet. My protocol is here for reference:

*All volumes used are 150 uL/well unless otherwise specified

*All washing steps were done by gently pipetting solution onto walls of each well instead of directly onto cells - this prevents cell detachment

*Antibody buffer for both primary and secondary ABs:

• SSC (20x)
• 2% goat serum
• 1% BSA
• 0.05% TritonX-100
• 0.02% NaN3
• dH2O

*AB wash solution:

• SSC (20x)
• 0.05% TritonX-100
• dH2O

1. After treatment of cells on microscope slides, wash cells 2x with PBS

2. Add 4% PFA (150 uL/well if 8-well slides are used)

• 4% PFA = 2g PFA + 50 mL PBS
• Make PFA fresh on day of

3. Let sit for 10 minutes

4. Remove PFA and wash 2x, 5 min each with glycine solution

• Glycine quenches the fixation

5. Remove glycine and wash 1x with PBS for 5 minutes

6. Add cold acetone for 10 min to permeabilize

7. Wash 3x with PBS (5 min each time)

8. After last PBS wash, remove PBS and block with antibody blocking solution for 30 min

• 10% donkey serum in PBS

9. Remove blocking solution and then add primary antibody (150 uL) in AB buffer overnight in 4 degrees

• 1:200 concentration for all ABs
• every well has VE-cadherin + one of the other TJ proteins; so two antibodies per well

10. The following day, remove primary Ab and wash 3x with antibody wash (10 minutes for each wash)

11. Add secondary and let sit for 1 hour

• I usually do two antibodies per well (VE-cadherin + one of the other tight junction proteins

12. Remove secondary and wash 3x (10 minutes each) with antibody wash

13. Wash 3x (5 minutes each) with PBS

14. Remove well dividers

15. Add prolong gold with DAPI (15 uL per well or 1 drop) to slide

16. Add cover slip to slide on top of prolong gold

• Make sure there are no air bubbles

17. Add nail polish around edges to seal edge

18. Image slides

Thanks in advance for reading and for the help!!