Hello,

I am studying cytokeratin 14 (K14) expression in Hacat human keratinocyte cell line and also von Willebrand factor (vWF) expression in choroid retinal endothelial cells (RF/6A). RF/6A cell line is obtained from rhesus species.

I have used following antibodies for the study:

1. Anti-cytokeratin 14 antibody (Anti-human) raised in mouse - ab7800 (Abcam)

2. Anti-vWF (anti-human with cross reactivity to non-human primates) Monoclonal Antibody (F8/86) raised in mouse- MA5-14029 (Thermofisher)

3. Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 633- A21052 (Thermofisher)

I have used following protocols for Immunofluorescence (IF) studies.

1. Culture respective cells on coverslip in sterile conditions overnight (until attachment occurs)

2. DPBS washes, fixation (two batches- 4% PFA and Methanol and DPBS washes again

4% PFA fixation: 20-25 mins at room temp

Methanol fixation: -20 deg Celsius for 8 mins

3. 1X DPBS washes (twice), Permeabilization in 0.2% and 1% Triton X 100 (Permeabilization was done only to RF/6A cell line samples fixed in 4% PFA to test for the presence of vWF)

4. Blocking - Conducted for 1h at room temp

a) For hacat, 5% BSA in DPBS was used as blocking agent

b) For RF/6A , two batches comprising of - 1% BSA only and 1.5%BSA+0.5% Triton X 100 were tested for blocking. (This was as per the protocols suggested by different colleagues in the institutes around ours).

5. Primary antibody dilutions:

a) Anti-cytokeratin 14- 1:50, 1:150, 1:250

b) Anti-vWF- 1:50, 1:100, 1:150

Incubation overnight at 4 degree Celsius in a humidified chamber.

6. 1X DPBS washes thrice on a shaker, and incubation with secondary antibody (dilution used 1:500) for 1-2 h (in dark)

7. 1X DPBS washes thrice on a shaker (in dark)

8. Mounting of slides on EverBrite Hardset Mouting Media with DAPI (Biotium) and stored at 4 deg Celsius prior to imaging by laser scanning confocal microscopy.

Lasers used: Excitation - 633 nm

Emission- 647 nm (Tried from 637 nm- 655 nm)

We tried adjusting laser power and intensities. However, we were receiving bright red fluorescence of the background instead of the cells. DAPI fluorescence was observed properly. Even in negative control slides (only secondary antibody), we could only see background fluorescence like the ones attached herewith (negative control images of both 4%PFA and methanol fixation are attached).

Thus, we are not able to study marker expression properly.

I have attached images of:

a) 4%PFA fixed negative control

b) Methanol fixed negative control

c) K14 4%PFA fixed 1:50 dilution (primary Ab)- Hacat cell line

d) K14 Methanol fixed 1:50 dilution (primary Ab)- - Hacat cell line

e) vWF (4% PFA fixative, 1.5%BSA+0.5%TritonX blocking and 1:50 primary Ab dilution) - RF/6A cell line

f) vWF (Methanol fixative, 1%BSA only blocking and 1:50 primary Ab dilution) - RF/6A cell line

Kindly guide me regarding the same.

Thank you.