Immunofluorescence

Shanshan Gao

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Immunofluorescence

SG Shanshan Gao

Last updated date: Jan 12, 2021 Views: 763 Forks: 0

An abbreviated version of this protocol was published in Science Advances in Jul, 2020

TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy

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Immuno Fluorescence Staining

Stage	Method	Preparations
1. Growth of Cells on Coverslips	Place a coverslip in the bottom of each well of a 6 well plate Remove lid and place both plate and lid in UV Cross-linking machine Sterilize using Maximum Crosslinkage setting Add cell suspension to each well and label plate Grow at 37°C for a min. of 4hrs (prefer overnight)
2. Treatment of Cells	Treat cells as needed
3. Fixing Cells	Aspirate off media Wash with 1x PBS (optional) Aspirate Add 1ml 3% Paraformaldehyde Incubate for 12-15mins at RT	Thaw and prepare paraformaldehyde in advance Coverslips can be stored O/N in 1xPBS
4. Permeabilize Cells	Aspirate Wash once with 1xPBS Aspirate Add 1ml of 0.5% Triton Buffer Incubate for 5mins at RT	Prepare 1° and 2° antibodies Dilute antibodies in 5% Goat Serum
5. Optional Blocking Step	Block in 5% Goat Serum for 20mins – 1hour	Optional step – may also use 10% FBS in PS to block
6. 1° Antibody	Aspirate Wash twice with 1xPBS Add 100-200ml of 1° Antibody directly to the center of the coverslip Incubate for 20mins at 37°C (or 1 hour at RT or overnight at 4°C)	Make a little extra antibody solution to ensure all wells get equal amounts
7. Wash and 2° Antibody	Aspirate Wash three times with 1xPBS with a few minutes incubation at RT in between. Aspirate Add 100-200ml of 2° Antibody directly to the coverslip Incubate for 20mins at 37°C	Most FITC and Rhodamine 2° Antibodies are diluted at 1:200 in 1xPBS
8. DAPI Staining	Aspirate Wash once with 1xPBS Add 200ml of diluted DAPI //或Hoechst Incubate at RT for 1 minute and aspirate Wash once with 1xPBS	Dilute DAPI in 1xPBS at 1:2000 Coverslips can be stored O/N in 1xPBS at this stage
9. Mounting Coverslips	Add a drop of Antifade solution to each slide Lift coverslips using a needle and gently drain off excess fluid Place coverslip on slide with cells facing down	Try to avoid bubbles
10. Fixing Coverslips onto Slide	Fix the coverslip to the slide by applying a drop of nail varnish at each corner of the coverslip

Recipes

3% Paraformaldehyde Solution

Per 500ml（多聚甲醛）

Warm 500ml 1x PBS in Microwave
Add 15g Paraformaldehyde
Shake for ~ 2mins
Add 2-5 drops 1M NaOH to dissolve paraformaldehyde
Add 10g Sucrose and shake until dissolved
Aliquot and Freeze
Store at -20°C

0.5% Triton Solution

Per 500ml

20mM Hepes pH7.4 (2.383g)
50mM NaCl (1.461g)
3mM MgCl₂ (304.95mg)
300mM Sucrose (58.85g)
Make up to 500ml
Filter solution

Anti Fade (light sensitive)抗淬灭

Per 50ml

Dissolve 5mg of p-Phenylenediamine (antibleach) in 5ml 1xPBS (warm to 37°C and vortex until dissolved)
Mix with 45ml Glycerol
Store at -20°C and wrap in foil

How to cite：

Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:

Gao, S(2021). Immunofluorescence. Bio-protocol Preprint. bio-protocol.org/prep744.
Gao, S., Guan, H., Yan, S., Hu, S., Song, M., Guo, Z., Xie, D., Liu, Y., Liu, X., Zhang, S. and Zhou, P.(2020). TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy . Science Advances 6(28). DOI: 10.1126/sciadv.aba7822