Cadherin Immunofluorescence

Cadherins are single chain glycoproteins involved in cell-cell adhesion. These junctions are calcium dependent. This protocol is adapted from Mike Bindschadler’s thesis.

Storage:

Store antibodies at 4C for 1-2 weeks; For long-term storage, aliquot and store @ -20 or -80C; Before aliquoting black bottom vials, place in Eppendorf tube and spiin @ 12000 rpm for 20 seconds.

Antibodies have been aliquoted and put in the -20C freezer (Feb 5 2009). Primary anti pan-cadherin aliquots are 4 uL and labeled “cad” for cadherin. Secondary TRITC aliquots are 20 uL and labeled “SaM” for sheep anti-mouse.

Antibodies from Abcam:

Primary Ab (ab6528) = monoclonal mouse anti pan-cadherin IgG, used @ 1:500 (references say 1:100-1:1000); 8.1 mg/mL in ascites fluid w/ 0.1% sodium azide

Secondary Ab (ab6805) = polyclonal TRITC (550nm/570nm) conjugated sheep anti-mouse IgG, used 1:50; 2 mg/mL in phosphate, sodium chloride, sodium azide, thimerisol, BSA buffer

Protocol:

Note: PBS is used at 1X concentration

1. Grow cells as normal on glass slides, wait for several days after confluence to allow junctions to form

2. Wash cells 1x in serum-free media and aspirate media

3. Fix cells in 4% PFA (in PBS) for 10 minutes at room temperature

4. Wash 1X in PBS and then soak in PBS 5 minutes

5. Permeabilize in 0.2% Triton X-100 for 5 minutes; wash

6. Block in 200 uL 4% BSA (in PBS) for 30 minutes in humidified chamber; aspirate

7. Add 200uL primary Ab (diluted 1:500 in 4% BSA) for 60 minutes in humidified chamber

8. Wash 3x in PBS (in different containers)

9. Add secondary antibody (diluted 1:200 in 4% BSA) for 60 minutes

10. Wash 3x in PBS, allowing cells to rest in final PBS wash for 5 minutes in humidified chamber

10a. This is a good time to counterstain with Hoechst 33342.  Let the cells rest in 1uM PBS for the final 5 minutes and then rinse again 1X with PBS

11. Clean excess fluid from coverslip, drop ~ 25 uL of mounting media on glass slide and mount coverslip on slide

12. Adhere coverslip to slide with clear nail polish and let set for 15 minutes

13. Observe!

Additional Information:

Although this didn’t seem to help, treating cells with sodium borohydride can reduce autofluorescence by a reduction mechanism.  Add these steps in between the fixation and permeabilization steps:

WARNING: Sodium borohydride is a highly flammable, toxic solid that is fatal is inhaled.  Contact with water (like in this protocol) releases flammable gas that may ignite spontaneously.  Weigh out and perform steps 5+6 in the fume hood.

1. During wash, prepare 1 mg/mL sodium borohydride (0.1%) in PBS; NaBH4 will foam after adding the PBS so use a big tube

2. Incubate samples 2x in 1 mg/mL NaBH4, 2 minutes each

3. Wash 1x in PBS and then soak in PBS, 5 min

Concentration Optimization:
The following was an experiment I did to try to find the optimal primary and secondary antibody concentrations for labeling cadherins.  These were P9 b.End3 cells grown on acid-washed glass coverslips within cloning rings, seeded at 50000 cells/cm2 and labeled 7 days post-seeding.  The primary antibody is a pan-cadherin and the secondary is TRITC-labeled.  I chose 3 primary and secondary antibody concentrations to span alomst the entire range of concentrations that I’ve seen used in the literature:

Most of these images had a significant amount of background fluorescence since the labeled proteins were not very bright.  Also keep in mind that these images have not been altered/adjusted with ImageJ or Photoshop for optimal presentation.  They are “as-taken”.  The background fluorescence seemed highest in the 1:250 and 1:750 primary images, and it increased with lower secondary antibody concentrations.  The most specific labeling of intercellular regions occured with the lower secondary antibody concentrations.  A 1:300 dilution of the secondary didn’t further increase the staining specificity (May 2009).  Empirically, I though that a 1:500 primary and 1:100 or 1:200 secondary concentration was optimal.

I also plotted the maximum brightness after adjusting the images with autoscale in ImageJ.

Surprisingly, the brightness did not increase dramatically with higher secondary antibody concentrations.  In fact, the maximum brightness for all the conentrations was about the same (there was a punctate bright spot in the 1:750, 1:50 image).  Since there is no brightness benefit to using higher concentrations of antibody, I feel that the proposed 1:500 primary and 1:100 or 1:200 secondary concentrations are appropriate.