Cy 3 affinipure f ab 2 fragment goat anti mouse igg h l

Anti-TERF1 antibody—ChIP Grade (ab1423) (Abcam, Cambridge, United Kingdom)—was used at 1:100 dilution for IHC, 1:300 for IF and 1:500 for WB. Anti-Myc-tag antibody [9E10]—ChIP Grade (ab32) (Abcam, Cambridge, UK)—was used at 1:500 dilution for WB and 1:500 for WB. Anti-TERF1-tsi (AB2) polyclonal antibody (Davids Biotechnologie GmbH, Regensburg, Germany) was used at 1:100, 1:200, and 1:400 for IHC, 1:100 for IF and 1:100, and 1:200 for WB. Anti-β-Actin monoclonal antibody produced in mouse (Sigma Aldrich, Missouri, USA) was used at 1:10,000 for WB. Anti-TBP antibody produced in goat (Sigma Aldrich, Missouri, USA) was used at 1:1000 in WB. Secondary antibodies: goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, California, USA) 1:2000 for IF. Cy™3 AffiniPure F(ab’)₂ fragment goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, Cambridgeshire, UK) was used at 1:1000 for IF.

Cells previously seeded on gelatin-coated coverslips were fixed with 3% paraformaldehyde for 10 min at room temperature, washed with PBS and permeabilized in 0.5% Triton X-100 in PBS for 4 minutes (min) on ice. A blocking step was performed by incubation with 1% Bovine Serum Albumin (BSA; Cat# 05470-5 G, Sigma-Aldrich) in PBS for 15 min and subsequently the cells were incubated with primary antibodies for OCT4 (monoclonal clone 7F9.2 m, Cat# MAB4419, Merck Millipore, 1:200 dilution), SSEA-1 (monoclonal clone MC-480, Cat# MAB4301, Merck Millipore, 1:100 dilution) and NANOG (polyclonal, Cat# RCAB002P-F, Reprocell, 1:150 dilution) diluted in 1% BSA/PBS for 45 min. After three washes with PBS, cells were incubated for 45 min with the secondary antibody Cy™3 AffiniPure F(ab’)₂ Fragment Goat Anti-Mouse IgG (H + L) (polyclonal, Cat# 115-166-003, Jackson ImmunoResearch Laboratories Inc., 1:200 dilution). 40,6-diamidino-2-phenylindole (DAPI, 0.2 mg/ml; Cat# D9542, Sigma) was used to stain the DNA and mark the nuclei by incubating at RT for 2 min. Cells were imaged using Zeiss Axio Observer (Carl Zeiss MicroImaging) with 63× oil objective using the filter sets FS43HE and FS49 and digital images were processed using FIJI platform (ImageJ v2.1.0/1.53q; [https://fiji.sc/]).