BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.
PBS (without Ca2+ and Mg2+)
Invitrogen
17-517Q
Optimal Cutting Temperature (OCT) Compound 4583
Tissue-Tek
4583
Superfrost Plus
Polysine slides
Menzel-Gläser, Germany
SF41296SP
Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.
Tween 20
Reagent
Calbiochem
655205
Tween 20 (0.5%), is a detergent to permeabilise cells
Triton X-100
Reagent
Sigma-Aldrich
T8787
Triton X-100 (0.1%) used to permeabilise cells.
Paraformaldehyde
Reagent
Sigma-Aldrich
158127
Anti-phospho-γH2AX (rabbit polyclonal antibody)
Primary Antibody
Upstate, USA
07-164
Dilution of primary antibody (1:500), in 1% BSA.
Alexa Fluor 488 goat anti-rabbit IgG (H+L)
Secondary Antibody
Invitrogen
A-11008
Dilution of secondary antibody (1:500), in 1% BSA.
Ethanol (70%)
Thermo Fisher
BSPEL316
RNAse A
Reagent
Qiagen
19101
Vectashield PI
Reagent
Vector Laboratories
H-1300
A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index.
Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.
Tang, M. M., Mah, L., Vasireddy, R. S., Georgiadis, G. T., El-Osta, A., Royce, S. G., Karagiannis, T. C. Quantitation of γH2AX Foci in Tissue Samples. J. Vis. Exp. (40), e2063, doi:10.3791/2063 (2010).