Confocal microscopy and immunofluorescence protocols, optimization methods and techniques - is there an immunofluorescence or confocal microscopy protocol you want to share? Do you have some comments, protocols or suggestions regarding deconvolution or scientific imaging tips? We want your feedback.

Here is a list of some commonly used solutions for immunofluoresence and immunohistochemistry.

1. PBS

a. 8 g NaCl
b. 0.2 g KCl
c. 1.44 g Na2HPO4
d. 0.24 g KH2PO4
e. Add 1 L distilled water
f. pH to 7.4

2. Paraformaldehyde - 4%

a. 8 g paraformaldehyde powder
b. 200 ml PBS (enough for 1 rack of microscope slides)
c. Stir solution and heat to 60 – 65 C (not higher!)
d. Adjust pH to 7.2
e. The solution will become clear once the paraformaldehyde has dissolved
f. Allow to cool. Use within a few days.

3. Permeabilization and blocking solution

a. Take a 15 mL tube and add 0.2 g BSA
b. Add 50 microlitres Triton X-100
c. Add 10 mL PBS.
d. Vortex until dissolved.
Use approximately 100 microlitres per tissue section

4. Mounting Medium

a. To a 50ml tube, add 6 g glycerol and 2.4 g mowiol.
b. Add 6ml distilled water, mix and leave for 2 hours RT (room temperature).
c. Add 12ml 0.2M Tris buffer solution (pH 8.5) and incubate at 50-55 C.
d. Stir occasionally to allow mowiol to dissolve.
e. Centrifuge – @ 5000 RPM for 20 min.
f. Aliquot and store at -20 C.
g. At RT, the solution is stable for 1 month.