Differentiated cultures were fixed with formaldehyde, followed by 45 min permeabilization in 0.5% triton X-100 and then blocked in 10% normal goat serum (DAKO) for 1 h. The corresponding primary antibodies for Nestin (Millipore, mouse-anti-Nestin [1:200]), Nanog (R&D System, goat-anti-Nanog [1:20]), Sox2 (R&D System, mouse -anti-Sox2 [1:50]), Oct4 (Abcam, rabbit-anti-Oct4 [1:200]), ß-Tubulin III (Millipore, mouse-anti-Tubulin [1:1000]), GFAP (Dako, rabbit-anti-GFAP [1:100]), S100 (Dako, rabbit-anti-Rabbit-S100 [1:250]), PGP9.5 (Dako, rabbit-anti-PGP9.5 [1:250]) and p75 (Abcam, mouse-anti-p75 [1:500]) were added and incubated over night at 4 °C. Samples were washed three times with PBS and incubated with the corresponding secondary antibodies (Alexa® 488 anti-mouse-IgG, Alexa® 488 anti-rabbit-IgG, Alexa® 488 anti-goat-IgG for 4 h at room temperature (RT). 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, 0.3 μg/mL) was used as nuclear stain. Cell numbers were related to an overall cell count based on DAPI stainings.
Mouse anti sox2
Manufactured by Abcam
Mouse anti-Sox2 is a primary antibody that specifically recognizes the Sox2 protein, a transcription factor involved in the regulation of embryonic development and the maintenance of stem cell pluripotency. This antibody can be used to detect and analyze the expression of Sox2 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Mouse anti lama c, by Santa Cruz Biotechnology (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Mouse anti α tubulin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti oct4, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Mouse anti p75, by Abcam (1 mentions)
Rabbit anti oct4, by Merck Group (1 mentions)
Rabbit anti hmgb1, by Cell Signaling Technology (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Ap detection kit, by Merck Group (1 mentions)
Diaminophenylindole dapi, by Merck Group (1 mentions)
Goat anti gata4, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nanog, by Santa Cruz Biotechnology (1 mentions)
Goat anti oct4, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse anti sox2
1
Immunocytochemical Characterization of Cultures
2
Immunoprecipitation of OCT4 Protein
3
Identification of OCT4 Interacting Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation was carried out as described.33 CGR8 cell lysates were harvested by Myc lysis buffer (138 mM NaCl, 20 mM Tris‐HCl (pH 8.0), 1% Nonidet P‐40), and anti‐OCT4 antibody or rabbit immunoglobulin G were used to immunoprecipitate OCT4 by incubating with protein A/G‐Magnetic Beads (Thermo Scientific) at 4 °C for 4 hours. After extensive washes, immunoprecipitated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membrane, immunostained with anti‐HMGB2 antibody, and finally detected by horseradish peroxidase‐conjugated secondary antibodies and visualized by chemiluminescence (Pierce). For whole‐cell lysate analysis, cells were resuspended in radio immunoprecipitation assay lysis buffer and total protein was quantified by the BCA protein assay kit (Pierce). Western blotting analysis was conducted with antibodies as follows: rabbit anti‐OCT4 (Abcam); mouse anti‐SOX2 (Abcam); rabbit anti‐HMGB2 (Cell Signaling Technology); rabbit anti‐HMGB1 (Cell Signaling Technology); mouse anti‐β‐ACTIN (Sigma); rabbit anti‐cyclic adenosine monophosphate response element‐binding protein (Cell Signaling Technology); mouse anti‐LamA/C (Santa Cruz); and mouse anti‐α‐Tubulin (Santa Cruz).
4
Immunophenotyping Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining assay was performed according to standard protocols. Briefly, the cells were fixed in 4% paraformaldehyde and washed with phosphate-buffered saline (PBS) (Invitrogen, 10010023) and then incubated in PBS containing 0.2% Triton X-100 (Sigma-Aldrich, 9002931) and 0.3% bovine serum albumin (Invitrogen, 11020021). Afterwards, the cells were incubated with primary antibodies including: goat anti-Oct4 (Abcam, ab27985), mouse anti-Nanog (Santa Cruz, sc-374001), mouse anti-Sox2 (Abcam, ab75485), goat anti-GATA4 (Santa Cruz, sc-1237), mouse anti-tyrosine hydroxylase (TH) (Abcam, ab6211), and goat anti- alpha fetoprotein (AFP) (Santa Cruz, sc-8108). The cells were then washed and incubated with goat anti-mouse IgG or rabbit-anti-goat IgG (Invitrogen). A concentration of 0.5 µg/ml diamino phenyl indole (DAPI) (Sigma, 28718903) was used for nuclei staining. Images were visualized using Nikon Ti inverted fluorescence microscope. Alkaline phosphatase (AP) staining was performed using the AP detection kit (Millipore, SCR004).
5
Immunohistochemical Analysis of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain sections were washed 4× 10 min in Tris-buffered saline (TBS) at room temperature, followed by a 1-h blocking step in TBS2+ (TBS with 0.3% horse serum [Millipore] and 0.3% Triton X-100 [Sigma]) at room temperature. The tissue was transferred to 0.5 mL Safe Lock Reaction Tubes containing 200 μL TBS2+ including primary antibodies. Samples were incubated for 24−48 h at 4°C. Tissue samples were washed 4× 10 min in TBS at room temperature, followed by a 30-min blocking step in TBS2+ at room temperature. Brain sections were transferred to 0.5 mL Safe Lock Reaction Tubes containing 200 μl TBS2+ including secondary antibodies. Samples were incubated in the dark for 2 h at room temperature. Subsequently, brain slices were washed 4× 10 min in TBS at room temperature and were mounted on glass slides with Fluoromount G (eBioscience). The following antibodies were used: mouse anti-Sox2 (Abcam; 1:100), guinea pig anti-DCX (Merck; 1:400), rabbit anti-S100B (Abcam; 1:100), goat anti-mCherry (SICGEN; 1:1,000), and chicken anti-GFAP (GeneTex; 1:500). Nuclei were counterstained with Hoechst 33342 (BioTrend; 1:3,000).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!