Double Immunofluorescence

General Protocol for double immunofluorescence with specific info on all antibodies that we have as of August 2010.

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Pan-cadherin Antibodies:

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

VEGF Antibodies:

Primary Ab (Abcam 1316) – mouse anti-mouse mAb for VEGF, aliquoted to 5uL in sterile HBSS and stored @-20C; used @ 1:100; this primary detects VEGF isoforms 121, 165, 189

Secondary Ab (R+D Systems NL009) – pAb NorthernLights 493nm (green) donkey anti-mouse IgG; 1 mg/mL in PBS with sodium azide; stored as-is @ 4C; used @ 1:500

Neurofilament H Antibodies:

Primary Ab (Cell Signaling Technology #2836) – mouse anti-mouse mAb for NF-H, 100uL stock in -20C freezer (do not aliquot); used @ 1:100

Secondary Ab (Abcam6805) – pAb TRITC (550nm/570nm) conjugated sheep anti-mouse IgG; 2 mg/mL in phosphate, sodium chloride, sodium azide, thimerisol, BSA buffer; 20uL aliquots in -20C freezer; used @ 1:500

ZO-1 Antibodies:

Primary Ab (ab59720) – polyclonal rabbit anti ZO-1 IgG, used @ 1:200; 0.1 mg/mL in 0.1% BSA, PBS and 0.02% sodium azide; Reacts with human, dog and mouse; I saw 1:200 as a reference

Secondary Ab (ab6717) – polyclonal FITC (495nm/528nm)-conjugated goat anti-rabbit IgG; 2 mg/mL in potassium phosphate, sodium chloride, 0.01% sodium azide, used @ 1:50

VEGFR2 Antibodies:

Primary Ab (R+D Systems MAB4432) – rat anti-mouse VEGFR2 mAb, 10uL aliquots of 500ug/mL stored @ -20C, use @ 1:50

Secondary Ab (R+D Systems NL015) – goat anti-rat NorthernLights 493nm (green), stored @ 4C, use @ 1:20 or 1:200; do NOT aliquot

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Solutions:

• 1X PBS, or other balanced salt solution
• 4% paraformaldehyde (PFA) or formaldehyde – toxic and carcinogenic so prepare and use in hood
• 0.3% Triton X-100 in 1X PBS
• Blocking buffer (1X PBS with final concentrations of 0.3% Triton X-100 and 4% BSA (or 5% normal serum from same species as the 2 antibody)
• Antibody dilution buffer (1X PBS with final concentrations of 4% BSA and 0.3% Triton X-100)
• 1X PBS, high salt (1X PBS, with 2.338g NaCl added per 100mL)
• ProLong Antifade Mounting Media (stored at -20C)

Protocol:

1. Culture cells & think about what surface you want to stain them on and how you’re going to keep the cells wet for an overnight incubation.
2. Rinse cells briefly in PBS.
3. Fix cells with 4% PFA for 15 minutes @ RT in the fume hood
4. Aspirate PFA and rinse cells 3X in PBS, 5 minutes each time.
5. Permeabilize cells in 0.3% Triton X-100 @ room temperature for ~15 minutes; IF NEEDED
6. Rinse cells 1X in PBS, 5 minutes.
7. Block cells in blocking buffer, 1-2 hours minutes at RT.
8. Make primary Ab solutions with antibody dilution buffer during the blocking step.
9. Aspirate blocking buffer and add primary Ab.  ~50uL per coverslip works but it will whisk off the coverslip onto whatever surface the coverslip is sitting on.  For cells in cloning/retention rings, 50uL per sample is a good volume.
10. Incubate overnight @ 4C in a humidified chamber.
11. Rinse 2X in PBS, 5 minutes; 1X in high salt PBS, 2 minutes and 1X in PBS, 5 minutes.
12. Make secondary Ab solutions in antibody dilution buffer.
13. Incubate cells in secondary Ab for 2 hours at RT.
14. Make 1uM Hoechst 33342 solution in 1X PBS (to counterstain nuclei) for next rinsing steps.
15. Rinse 2X PBS, 5 minutes; 1X in high salt PBS, 2 minutes and 1X in Hoechst-supplemented PBS, 15 minutes.
16. Mount on glass slides with ~ 15uL antifade mounting media per coverslip or add a 10uL drop of mounting media to cloning ring before observation.
17. Observe.