Immuno-fluorescent (IF) staining

Fluorescent IHC, Basic protocol

1. Fix the samples in 4% PFA in 1x PBS, pH 7.4 (see notes below)
2. Wash 1x 15 min in PBS to remove PFA
3. Postfix in 100% cold Methanol for 10 min at -20°C
4. Step into 0.1% triton x100 / PBS 
5. Permealize membranes in 0.5% triton X100 / PBS for 30 min at RT
6. To quench autofluorescence incubate in 0.1M glycine in 0.1% triton X100 / PBS for 30 min at RT
7. Wash 3 x15 min in 0.1% triton X100 / PBS
8. Incubate the samples with Blocking Solution (3% BSA in 0.1% triton X 100 / PBS) for 1h at RT
9. Prepare the 1^st antibody
10. Vortex and centrifuge Ab stocks in their original vials at 14000 rcf for 5 min. 

Dilute the 1^st antibody in Blocking Solution (if applicable). 

1. Apply the 1^st antibodies overnight at 4°C.
2. Wash  6 x15 min in Blocking Solution at RT
3. Prepare the 2^nd antibody:

Vortex and centrifuge Ab stocks in their original vials at 14000 rcf for 5 min

Dilute the 2^nd antibody in Blocking Solution

1. Apply the 2^nd antibody for 1h at RT
2. Wash 3 x 15 min in Blocking Solution
3. Wash 3 x 15 min in 0.1% triton X100 / PBS
4. Nuclear staining with DAPI – 1:10000 in PBS for 30 min at RT
5. Wash 3 x 5 min in PBS
6. Remove the excess of PBS and add the antifading medium.
7. Image!

Notes:

1. The fixative should be freshly prepared before use. Can be stored for 1 week at 4ºC.

Fixation time can vary from overnight incubation at +4°C to 15 min – 2h at RT.

PFA: Paraformaldehyde

2. 3-5% normal goat serum solution in 0.1 % triton X 100 / PBS can be use once at step 8 to block non-specific binding from the goat 2^nd antibody.

Antifading medium recipe:

DABCO (Aldrich, D2522) – 2.5%

Glycerol – 25%

in 0.2 M Tris-Hcl , pH 8.5

Notes specific to request:  Reduce background by 1. Proper fixation - over fixation can lead to background.  2. Correct dilution of antibody.  In the first experiment prepare a titration of antibody concentrations to identify the optimum signal to background.  3. Carefully rotate samples while washing after antibody treatment and ensure good wash exchanges (i.e. wash in 1 ml and remove all but 50 to 100ul of solution after each wash)