Fix the samples in 4% PFA in 1x PBS, pH 7.4 (see notes below)
Wash 1x 15 min in PBS to remove PFA
Postfix in 100% cold Methanol for 10 min at -20°C
Step into 0.1% triton x100 / PBS
Permealize membranes in 0.5% triton X100 / PBS for 30 min at RT
To quench autofluorescence incubate in 0.1M glycine in 0.1% triton X100 / PBS for 30 min at RT
Wash 3 x15 min in 0.1% triton X100 / PBS
Incubate the samples with Blocking Solution (3% BSA in 0.1% triton X 100 / PBS) for 1h at RT
Prepare the 1st antibody
Vortex and centrifuge Ab stocks in their original vials at 14000 rcf for 5 min.
Dilute the 1st antibody in Blocking Solution (if applicable).
Apply the 1st antibodies overnight at 4°C.
Wash 6 x15 min in Blocking Solution at RT
Prepare the 2nd antibody:
Vortex and centrifuge Ab stocks in their original vials at 14000 rcf for 5 min
Dilute the 2nd antibody in Blocking Solution
Apply the 2nd antibody for 1h at RT
Wash 3 x 15 min in Blocking Solution
Wash 3 x 15 min in 0.1% triton X100 / PBS
Nuclear staining with DAPI – 1:10000 in PBS for 30 min at RT
Wash 3 x 5 min in PBS
Remove the excess of PBS and add the antifading medium.
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 2nd 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)
Copyright: Content may be subjected to copyright.
How to cite:
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
Zheng, M and F Hinman, V(2022). Immuno-fluorescent (IF) staining. Bio-protocol Preprint. bio-protocol.org/prep1953.
Zheng, M., Zueva, O. and Hinman, V. F.(2022). Regeneration of the larval sea star nervous system by wounding induced respecification to the Sox2 lineage. eLife. DOI: 10.7554/eLife.72983
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