Immunohistochemistry was performed on 50 μm tissue sections as previously described (Moldrich et al., 2010 (link)). Primary antibodies used: sheep anti-DRAXIN (1:250; AF6149, R&D Systems), mouse anti-human KI67 (1:500; 550609, BD Pharmingen), mouse anti-GAP43 (1:500; MAB347, Millipore), rabbit anti-GFAP (1:500; Z0334, Dako), mouse anti-GLAST (or EAAT1; 1:500; ab49643, Abcam), rabbit anti-GLAST (or EAAT1; 1:250; ab416, Abcam), chicken anti-LAMININ (1:250; LS-C96142, LSBio), rabbit anti-LAMININ (1:250; L9393, Sigma), rat anti-NESTIN (AB 2235915, DSHB), and rabbit anti-SOX9 (1:500, AB553, Merck). Secondary antibodies were Alexa Fluor IgG antibodies (1:500, Invitrogen) or biotinylated IgG antibodies (1:500 or 1:1000, Jackson Laboratories) used in conjunction with Alexa Fluor 647-conjugated streptavidin (1:500, Invitrogen) amplification. EdU labelling was performed using the Click-iT EdU Alexa Fluor 488 or Alexa Fluor 555 Imaging kits (Invitrogen) according to the manufacturer’s instructions. Cell nuclei were labelled using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen) and coverslipped using ProLong Gold anti-fade reagent (Invitrogen) as mounting media.
Ab553
Manufactured by Merck Group
AB553 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, providing basic functionalities for various scientific applications. The core function of AB553 is to facilitate standardized measurement and analysis procedures in a laboratory setting. No further details or interpretation about its intended use are available.
Automatically generated - may contain errors
Lab products found in correlation
Z0334, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Click it edu alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Mab347, by Merck Group (1 mentions)
L9393, by Merck Group (1 mentions)
Ab416, by Abcam (1 mentions)
Ls c96142, by LSBio (1 mentions)
Af6149, by R&D Systems (1 mentions)
T9026, by Merck Group (1 mentions)
Protein dye reagent, by Bio-Rad (1 mentions)
A5316, by Merck Group (1 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Ab24834, by Abcam (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using ab553
1
Immunohistochemistry Protocol for Tissue Analysis
2
Protein Extraction and Immunoblotting Protocol
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All cell lysis was performed with RIPA buffer (Cell Signaling Technology) with added cOmplete protease inhibitor cocktail (25× dilution; Roche), 1 mmol/L phenylmethylsulfonyl fluoride (Sigma-Aldrich), and phosphatase inhibitor cocktails 2 and 3 (100× dilution; Sigma-Aldrich). Total protein extracted was quantified by the Bradford assay with the Bio-Rad protein dye reagent (Bio-Rad). Quantified protein samples, denatured by heating, were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Merck Millipore). Primary antibodies used for immunoblotting against various targets were listed as follows: PTK7 (1:1000, AF4499; R&D Systems), SOX9 (1:1000, AB553; Merck Millipore), Smad2 (1:1000, 5339; Cell Signaling Technology), pSmad2 (1:1000, 3108; Cell Signaling Technology), SLUG (1:1000, MA5-26385; Thermo Scientific), ZEB1 (1:500, ab203829; Abcam), β-catenin (1:1000, 9587; Cell Signaling Technology), α-tubulin (1:1000, T9026; Sigma-Aldrich), histone H3 (1:2000, ab24834; Abcam), and β-actin (1:10,000, A5316; Sigma-Aldrich).
3
Immunostaining of Neuronal Markers
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(1:250, ab610921, BD Biosciences), rat anti-Nestin (AB 2235915, DSHB), chicken anti-Nestin (1:1000, ab134017, Abcam), goat anti-Netrin1 (AF1109, R&D Systems), mouse anti-Neurofilament (1:500; MAB1621, Chemicon), rabbit anti-Nfia (1:500; ARP32714, Aviva Systems Biology), rabbit anti-Nfib (1:500; HPA003956, Sigma), rabbit anti-neuronal-specific-ßIII-tubulin (1:500; ab18207, Abcam), rabbit antiphospho p44/42 Mapk (or Erk1/2; 1:250; 9101, Cell Signaling), rabbit anti-Sox9
(1:500, AB553, Merck), and goat anti-TDTOMATO (1:500, ab8181-200, Sicgen). For actin staining, Alexa fluor-conjugated phalloidin (A22287, Thermofisher scientific) was incubated on tissue for thirty minutes in the dark as per the manufacturer's instructions, prior to the addition of primary antibodies. Immunohistochemistry was performed in a similar manner for cultured cells, with the following minor exceptions: HEK293T cells expressing wildtype and mutant pCAG-DCC:TDTOMATO constructs were not permeabilized to confirm exogenous DCC receptor localisation to the plasma membrane.
(1:500, AB553, Merck), and goat anti-TDTOMATO (1:500, ab8181-200, Sicgen). For actin staining, Alexa fluor-conjugated phalloidin (A22287, Thermofisher scientific) was incubated on tissue for thirty minutes in the dark as per the manufacturer's instructions, prior to the addition of primary antibodies. Immunohistochemistry was performed in a similar manner for cultured cells, with the following minor exceptions: HEK293T cells expressing wildtype and mutant pCAG-DCC:TDTOMATO constructs were not permeabilized to confirm exogenous DCC receptor localisation to the plasma membrane.
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