Hola Alvaro, some of my colleagues used in the past the abcam ki67 clone[SP6] (ab16667) antibody with an antigen retrieval protocol (10mM citrate buffer/EDTA 1mM/Tween 0.05% pH 6.0 ) x 30 min @ 90°C.

Ab dilution 1/1000

Suerte

I don´t have this ab. I have an ab from BD and other from Santa Cruz. I think that I can apply a similar protocol with antigen retrieval. 

And one question more. What kind of fixation that you use?

Gracias

Extra links

https://www.researchgate.net/post/Ki67_immunostaining

https://www.google.de/search?client=tablet-android-lenovo&q=ki67+staining+protocol&sa=X&ved=0ahUKEwi0s7m8joHSAhXBPxQKHVwzBcEQ1QIIdSgA&biw=1280&bih=800

 thanks 

Fixation:4% PFA

Antigen retrieval: sodium citrate

detergent:Triton/TBs

Incubation: for sure should be 4 degrees overnight

Concentration in santacruz:1:300

Hi Alvaro,

I have performed Ki67 staining in neonatal and adult mouse brain.

Neonatal brains were drop fixed in 4% PFA overnight. Adult brains were perfused with RPMI+6 units/ml heparin followed with 4% PFA and drop fixed in 4% PFA overnight. Then, the brains were cryoprotected overnight with 30% sucrose and frozen in OCT the next day.

I worked with 40 micron thaw-mounted sections. Antigen retrieval was not necessary, but I did perform a 30 minute wash/perm step with 1% Triton-X100 in TBS followed by two 5 min washes with TBS, blocking with a TBS-based superblock+0.3% Triton-X100, and then overnight incubation with primary in diluent (1:5 dilution of TBS-based superblock+0.3% Triton-X100). The following antibodies have worked beautifully in my mouse brain tissues:

rabbit anti-Ki67 (Vector, Burlingame, CA, USA, www.vectorlabs.com; Z0311, 1:500)

rat anti-Ki67 (eBioscience, Waltham, MA, USA, www.ebioscience.com; 14-5698-82, 1:200)

Good luck!

Steph

Thank you for your answers