To perform immunocytochemistry, cells were fixated with 4% paraformaldehyde and blocked with blocking buffer containing 5% normal goat serum (Gibco®), 0.1% bovine serum albumin (SigmaAldrich) and 0.3% Triton X-100 (SigmaAldrich). Primary antibody incubation for MeCP2 (D4F3, CellSignaling, 1:200, rabbit), OCT3/4 (C-10, Santa Cruz, 1:1000, mouse), SSEA4 (Developmental Studies Hybridoma Bank, 1:50, mouse), TRA1-60 (Santa Cruz, 1:200, mouse), TRA1-81 (Millipore, 1:250, mouse) and SOX2 (Millipore, 1:1000, rabbit) was performed in blocking buffer over night at 4 °C. Next day, cells were washed, and secondary antibody Alexa Fluor® 488 (ThermoFisher, 1:1000, mouse or rabbit) and Alexa Fluor® 594 (ThermoFisher, 1:1000, mouse or rabbit) were applied in blocking buffer for 1 h at room temperature. To identify cell nuclei, DAPI was used for 5 min before cells were mounted with Fluoromount™ (Sigma-Aldrich).
Oct3 4 c 10
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
OCT3/4 (C-10) is a laboratory product used for research purposes. It is a monoclonal antibody that specifically recognizes the OCT3/4 protein, which is a transcription factor involved in the regulation of embryonic stem cell pluripotency and self-renewal. The antibody can be used in various experimental techniques, such as immunohistochemistry, immunocytochemistry, and Western blotting, to detect and analyze the expression of OCT3/4 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (3 mentions)
Tra 1 60, by Santa Cruz Biotechnology (3 mentions)
Tra 1 81, by Merck Group (3 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Fluoromount, by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Ssea4, by Developmental Studies Hybridoma Bank (3 mentions)
N cadherin clone 3b9, by Thermo Fisher Scientific (1 mentions)
Decma 1, by Santa Cruz Biotechnology (1 mentions)
Mec 13, by Santa Cruz Biotechnology (1 mentions)
Tra 1 60, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using oct3 4 c 10
1
Immunocytochemistry Assay for Pluripotency Markers
2
Immunocytochemistry Protocol for Cell Markers
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For immunocytochemistry, the following antisera were used: rat monoclonals against VE-cadherin (11D4.1, BD Biosciences), PECAM-1 (MEC 13.3, Santa Cruz), and E-Cadherin (DECMA-1, Santa Cruz), mouse monoclonals against cardiac Troponin T (CT3, Iowa Hybridoma Bank), Isl1 (39.4D5, Iowa Hybridoma Bank), Oct3/4 (C-10, Santa Cruz), SMA (Neomarkers), N-cadherin (clone 3B9, Invitrogen), MyHC (MF20, Iowa Hybridoma Bank), and a-actinin (Clone BM-75.2, Sigma), goat polyclonals against GATA4 (C-20, Santa Cruz) and Isl1 (GT15051-100, Acris Antibodies), rabbit monoclonals against MEF2c (D80C1) and VEGF receptor 2 (Flk1) (55B11) from Cell Signaling, and rabbit polyclonals against EGFP (kindly provided from Dr. Charalambia Boleti, Pasteur Institute, Athens), Desmoplakin 1/2 [27 (link)], and DSC2 (DSC2, RDI Research Diagnostics, Inc.).
3
Immunocytochemistry of Stem Cell Markers
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To perform immunocytochemistry, cells were fixated with 4% Paraformaldehyde and blocked with blocking buffer containing 5% Normal Goat Serum (Gibco®), 0.1% bovine serum albumin (SigmaAldrich) and 0.3% Triton X-100 (SigmaAldrich). Primary antibody incubation for MeCP2 (D4F3, CellSignaling, 1:200, rabbit), OCT3/4 (C-10, Santa Cruz, 1:1000, mouse), SSEA4 (Developmental Studies Hybridoma Bank, 1:50, mouse), TRA1-60 (Santa Cruz, 1:200, mouse), TRA1-81 (Millipore, 1:250, mouse), SOX2 (Millipore, 1:1000, rabbit) was performed in blocking buffer over night at 4°C. Next day cells were washed and secondary antibody Alexa Fluor® 488 (ThermoFisher, 1:1000, mouse or rabbit) and Alexa Fluor® 594 (ThermoFisher, 1:1000, mouse or rabbit) were applied in blocking buffer for 1 h at room temperature. To identify cell nuclei DAPI was used for 5 min before cells were mounted with Fluoromount™ (Sigma-Aldrich).
4
Immunocytochemical Characterization of Stem Cells
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To perform immunocytochemistry, cells were xated with 4% Paraformaldehyde and blocked with blocking buffer containing 5% Normal Goat Serum (Gibco®), 0.1% bovine serum albumin (SigmaAldrich) and 0.3% Triton X-100 (SigmaAldrich). Primary antibody incubation for MeCP2 (D4F3, CellSignaling, 1:200, rabbit), OCT3/4 (C-10, Santa Cruz, 1:1000, mouse), SSEA4 (Developmental Studies Hybridoma Bank, 1:50, mouse), TRA1-60 (Santa Cruz, 1:200, mouse), TRA1-81 (Millipore, 1:250, mouse), SOX2 (Millipore, 1:1000, rabbit) was performed in blocking buffer over night at 4 °C. Next day cells were washed and secondary antibody Alexa Fluor® 488 (ThermoFisher, 1:1000, mouse or rabbit) and Alexa Fluor® 594 (ThermoFisher, 1:1000, mouse or rabbit) were applied in blocking buffer for 1 h at room temperature. To identify cell nuclei DAPI was used for 5 min before cells were mounted with Fluoromount™ (Sigma-Aldrich).
5
Pluripotency Marker Expression Analysis
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Immunocytochemistry was performed using OCT3/4 (C-10, Santa Cruz Biotechnology, Santa Cruz, CA), TRA-1-60 (Abcam, Cambridge, UK), and PMEL (Abcam). Cells were then immunostained with isotype-specific secondary antibodies (Alexa-Fluor, Life Technologies). Nuclei were counterstained using DAPI (Sigma-Aldrich).
6
Agar-based Cell Block Preparation and Immunostaining
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The AgarCyto cell block preparation, immunostaining, and subsequent histopathological evaluation were described in detail elsewhere (13) . In summary, the remaining biopsy particles were fixed in a 4% formaldehyde fixative (Unifix, Klinipath) and transferred to the pathology laboratory. An agar solution (Agar technological no. 3, Oxoid Ltd.) was used to solidify the fixed cell suspension. After cytocentrifugation and solidification, the cone was halved and embedded in paraffin together with the standard testicular biopsy. Of each paraffin block 4-mm sections were cut at two levels. From each level sections were routinely stained with hematoxylin-eosin (H & E) and Elastin according to Masson for cytological examination. Immunohistochemistry was performed for screening of GCNIS, as markers two monoclonal antibodies were used: PLAP (PL8/F6, BioGenex) and OCT3/4 (C-10, Santa Cruz Biotechnology Inc.). A positive control (seminoma with GCNIS) was added to all slides. The GCNIS was identified by an experienced pathologist on scanning microscopy level by membranous staining of PLAP and nuclear staining of OCT3/4 in the same way as the control specimen on that slide.
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