Paraffin embedded tumor sections were deparaffinized by successive incubations in xylene, graded washes in ethanol, and PBS. Epitope unmasking was performed by steaming in 0.01 mol/L citrate buffer (pH 6.0) for 45 minutes. Paraffin embedded sections were immunostained with MYC (1:150, Epitomics), or cleaved capsase 3 (1:100, Cell Signaling technology), phospho histone 3 (1:200, Cell Signaling Technology), CD4 (1:1000, Abcam) and F4/80 ( 1: 150, Life technologies) overnight at 4oC. The tissue was washed with PBS and incubated with biotinylated anti-rabbit or anti-mouse for 30 minutes at room temperature (1:300 Vectastain ABC kit, Vector Labs). Sections were developed using 3,3’- Diaminobenzidine (DAB), counterstained with hematoxylin, and mounted with permount. Images were obtained on a Nikon microscope.
Cleaved capsase 3
Manufactured by Cell Signaling Technology
Cleaved caspase 3 is a protein that is a marker for apoptosis, or programmed cell death. It is a key executioner of apoptosis, as it is responsible for the proteolytic cleavage of many key cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain abc kit, by Vector Laboratories (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Phospho histone 3, by Cell Signaling Technology (1 mentions)
Novex ecl chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Xrcc4, by Santa Cruz Biotechnology (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rpa32, by Fortis Life Sciences (1 mentions)
P rpa32, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
3 protocols using cleaved capsase 3
1
Immunohistochemical Analysis of Tumor Markers
2
Radiation-Induced Apoptosis and DNA Repair Signaling
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Cells were irradiated at 70–90% confluence, and lysates were prepared at specific time points post-radiation injury. Cells were washed three times with PBS and then extracted with RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate and ×1 Halt Protease and phosphatase inhibitor (cat# 78443, Thermofisher, Rockville MD). Primary antibodies were obtained for full-length caspase 3 (cat. #9662S, Cell Signaling,, Danvers, MA) (1:1000), cleaved capsase 3 (cat. #9661S, Cell Signaling) (1:1000), and p21 (cat. #9sc-397, Santa Cruz Biotechnology, Inc., Dallas TX) (1:500), p-RPA32 (cat. #A300–245A, Bethyl, Montgomery, TX) (1:500), RPA32 (cat#A300–244A, Bethyl) (1:500), Rad51 (cat. #98875S, Cell Signaling) (1:500), Ku70 (cat. #9sc-9033, Santa Cruz Biotechnology) (1:1000), and XRCC4 (cat. #sc-8285, Santa Cruz Biotechnology) (1:300). Proteins were detected with species-matched horseradish peroxidase–linked secondary antibodies (1:500–1:2000, R&D Systems) and Novex ECL Chemiluminescent Substrate (Cat # WP20005, Thermofisher). WCIF ImageJ software was used for densitometry analysis (NIH, Bethesda MD; https://imagej.nih.gov/ij/download.html ).
3
Cryosectioning and Immunostaining of Cerebral Organoids
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For cryosectioning, fixed organoids were incubated in sucrose solution overnight at 4 °C before embedding and freezing in O.C.T medium. Frozen organoid tissue was sliced into 20 μm sections using a cryostat. Permeabilization/blocking was performed using 3% BSA/0.1% TX100 in TBS. Incubation of sections from cerebral organoids in primary antibodies solution was performed overnight at 4 °C and in secondary antibodies solution at room temperature for 1 hr (Alexa Fluor, Molecular Probes). The following primary antibodies were used: DCX (Aves Labs, 1:200); PAX6 (Millipore; 1:200); TBR1 (Abcam 1:400); Cleaved capsase-3 (Cell Signaling, 1:500). Coverslips were affixed with ProLong Gold antifade reagent with DAPI (Life Technologies) and z-stacks were acquired using either a Leica TCS SP8 confocal microscope.
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