The immunohistochemistry was performed using the Fast ImmunoCytoChemistry® Staining Kit (Protein Biotechnologies). Anti-FoxG1 antibody (Abcam, ab18259), anti-LC3B antibody (Sigma-Aldrich, L7543), anti-Myosin7a antibody (Proteus Bioscience, 25–6790), a TUNEL Kit (Roche, 11684817910), and DAPI were used for analyzing FoxG1 expression and detecting autophagy, apoptotic cells, and nuclei, respectively.
Ab18259
Manufactured by Merck Group
Ab18259 is a lab equipment product from Merck Group. It is designed to perform specialized tasks in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3b antibody, by Merck Group (1 mentions)
Tunel kit, by Roche (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Rhodamine phalloidin, by Yeasen (1 mentions)
Ab92391, by Abcam (1 mentions)
Af3369, by BioLegend (1 mentions)
Af3075, by Novus Biologicals (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dm6000 inverted microscope, by Leica (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Ab185050, by Abcam (1 mentions)
Ab176882, by Abcam (1 mentions)
L7543, by Abcam (1 mentions)
Ar1177, by Boster Bio (1 mentions)
Mab5326, by BioLegend (1 mentions)
A2052, by Merck Group (1 mentions)
Abn78, by Merck Group (1 mentions)
Quickblock blocking buffer for immunol staining, by Beyotime (1 mentions)
6 protocols using ab18259
1
Immunohistochemical Analysis of Cellular Markers
2
Comprehensive Subcellular Profiling of FOXG1
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Anti-FOXG1 antibody (Abcam, ab18259), anti-LC3B antibody (Sigma-Aldrich, L7543), anti-H3K9me2 antibody (Abcam, ab176882), Moysin7a (Proteus Biosciences, 25-6790), rhodamine phalloidin (Yeasen), and DAPI (Solarbio) were used to analyze the FOXG1 expression and detect autophagy, H3K9me2, HCs, and microfilament structure and nucleus, respectively.
The samples were incubated in 4% paraformaldehyde (Sigma-Aldrich) for 1 h and then blocked with 0.5% Triton X-100 (blocking medium) for 1 h. The primary antibodies were then added at a 1:400–1:1,000 dilution and incubated overnight at 4°C. The samples were washed thrice with PBST, incubated with fluorescent secondary antibodies for 1 h at room temperature in the dark, rewashed thrice with PBST, and reincubated with rhodamine phalloidin and DAPI for 30 min in the dark. After sealing the slides with clear nail polish, we imaged them using a confocal microscope.
The samples were incubated in 4% paraformaldehyde (Sigma-Aldrich) for 1 h and then blocked with 0.5% Triton X-100 (blocking medium) for 1 h. The primary antibodies were then added at a 1:400–1:1,000 dilution and incubated overnight at 4°C. The samples were washed thrice with PBST, incubated with fluorescent secondary antibodies for 1 h at room temperature in the dark, rewashed thrice with PBST, and reincubated with rhodamine phalloidin and DAPI for 30 min in the dark. After sealing the slides with clear nail polish, we imaged them using a confocal microscope.
3
Immunocytochemistry of Neural Stem Cells
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Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were blocked with 10% donkey serum in PBS for at least 1 h. Primary antibodies against Nestin (Abcam, ab92391), SOX2 (Abcam, ab59776), SOX1 (R&D Systems, AF3369), PAX6 (BioLegend, PRB-278P), Ki67 (CST, 9449S), Dlx2 (Abcam, ab117546), FOXG1 (Abcam, ab18259), MAP2 (Sigma Aldrich M9942), TBR1 (Abcam ab31940), BRN2 (Cell Signaling 12,137), NeuN (Abcam ab104225), SOX9 (R&D Systems AF3075), GLT1 (Novus Biologicals NBP1–20136), and GFAP (Dako Z0334) were diluted in blocking buffer and incubated overnight at 4 °C. Following several washes, the cells were incubated with the appropriate donkey anti-rabbit IgG, anti-mouse IgG, or anti-goat conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 secondary antibodies (Life Technologies) for 1 h at room temperature. Cells were counterstained with DAPI and visualized with the Leica DM6000 inverted microscope. Images were acquired using the Q-Imaging Retiga Xi Firewire High-Speed, 12-bit cooled CCD camera and Volocity software. Staining was quantified using ImageJ software and represented as a portion of total nuclei. On average, 2.5 × 103 cells were quantified from each experiment. Results are shown with the standard deviation of the mean of three independent experiments.
4
Western Blot Analysis of Protein Markers
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SDS-PAGE gel electrophoresis was performed to separate the proteins. The proteins on the gel were transferred to PVDF membranes, which were blocked with 5% nonfat milk in TBST for 1 h at room temperature on a shaker. The membrane was placed in the primary antibody, incubated overnight using a refrigerator shaker, washed three times with TBST for 5 min each, and then incubated with a 1:5,000 secondary antibody for 1 h at room temperature. Washing with TBST was performed thrice for 5 min each, and exposure to film was achieved using an ECL solution in a dark room. The films were developed, fixed, and air dried. After scanning the films, we analyzed the immunoblot bands using Image J. The primary antibodies used were anti-FOXG1 antibody (Abcam, ab18259), anti-LC3B antibody (Sigma-Aldrich, L7543), anti-G9a antibody (Abcam, ab185050), and anti-H3K9me2 antibody (Abcam, ab176882).
5
Immunofluorescence Staining of Neural Cells
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Immunofluorescence staining was performed in vitro on coverslip cultures or frozen brain tissue sections as previously described [27 (link)]. Briefly, cells were fixed in 4% PFA for 20 min (frozen tissue sections did not require this step), washed with phosphate-buffered saline (PBS), and then blocked by QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, P0260) for 30 min at room temperature. The primary antibodies were added and incubated overnight at 4°C. Primary antibodies included mouse anti-Nestin (Millipore, MAB5326, 1 : 1000), rabbit anti-PAX6 (BioLegend, 901301, 1 : 1000), goat anti-SOX1 (R&D Systems, AF3369, 1 : 1000), rat anti-HOXB4 (DSHB, RRID: AB-2119288, 1 : 100), rabbit anti-Foxg1 (Abcam, Ab18259, 1 : 500), mouse anti-NKX2.1 (Millipore, MAB5460, 1 : 500), rabbit anti-GABA (Sigma-Aldrich, A2052, 1 : 1000), goat anti-CHAT (Millipore, AB144P, 1 : 300), mouse anti-neuronal class III β-tubulin (Tuj1; BioLegend, 801201, 1 : 2000), mouse anti-microtubule-associated protein-2 (Map2; Sigma-Aldrich, M1406, 1 : 1000), mouse anti-human nuclei (HuNu; Millipore, MAB1281, 1 : 500), goat anti-mCherry (Biorbyt, RRID: AB-2687829), and rabbit anti-NeuN (Millipore, ABN78, 1 : 1000). The corresponding fluorescent-conjugated secondary antibodies were applied for 1 h, and the nuclei were stained with DAPI (Boster, AR1177).
6
Immunostaining of Cultured Cells
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