Axioscan 7 microscope

Methyl group of O6-mG can be transferred to cysteine 145 (C145) of MGMT. When mutating this residue into serine, the resultant MGMT C145S mutant is still able to recognize O6-mG, but fails to remove methyl group of O6-mG, resulting in its retention on the DNA regions containing O6-mG. Based on this, we purified MGMT C145S mutant tagged with Flag as a “sticky” probe for detecting O6mG levels in xenograft tissues. After tumor cell implantation, the brain was isolated and sequentially dehydrated with 15% and 30% sucrose, respectively, before embedding in OCT and frozen on dry ice. OCT molds were sectioned at 10 μm thickness. Sections were washed with PBS and incubated in 0.1 mM of Flag-MGMT C145S recombinant protein for 2 h at 37 °C, followed by incubation with formaldehyde for 5 min. Sections were then washed with PBS, incubated in PBS with 3% bovine serum albumin (BSA) at room temperature for 1 h and incubated overnight at 4 °C in primary antibody against Flag. The following day, sections were washed three times in PBS, incubated with a secondary antibody against the appropriate species (1:500) diluted in PBS with 1.5% BSA at room temperature for 1 h, and washed three times in PBS. Cell nuclei were stained using DAPI and fluorescence images were captured using a ZEISS AxioScan7 microscope.

10.17504/protocols.io.5jyl89m9dv2w/v1
Slides were de-paraffinized with 2 sequential 5 min washes in xylenes, followed by 1 min washes in a descending series of ethanols: 100%, 100%, 95%, 80%, 70%. Slides were then incubated in deionized water for one min prior and transferred to the BioGenex EZ-Retriever System where they were incubated in antigen unmasking solution (Vector Laboratories; Cat# H-3300) and microwaved for 15 min at 95 °C. Slides were allowed to cool for 20 min at room temperature and washed in running tap water for 10 min. Slides were washed for 5 min in 0.1 M Tris, then blocked in 0.1 M Tris/2% fetal bovine serum (FBS) for 2 h. Slides were incubated in primary antibody in 0.1 M Tris/2% FBS in a humidified chamber overnight at 4 °C.
Primary antibodies were rinsed off with 0.1 M tris and incubated in 0.1 M Tris/2% FBS for 5 min. Slides were then incubated in the dark for 3 h at room temperature with secondary antibodies in 0.1 M Tris/2% FBS. Slides were rinsed three times for 10 min in 0.1 M Tris in the dark, then mounted with coverglass in ProLong gold with DAPI (Invitrogen, Cat#P36931). Fluorescent slides were imaged at 20X magnification on a Zeiss AxioScan 7 microscope.