Mice were anesthetized and perfused with normal saline (NS) and 4% paraformaldehyde. After that, the brain was rapidly taken out and placed in 4% paraformaldehyde for 2 days followed by paraffin embedding and sectioning. After continuous rehydration with 30% sucrose solution and washing with PBS, endogenous peroxidase activity in the tissue sections was blocked by 3% H2O2 for 25 min at room temperature. Antigen crosslinking was then conducted followed by two PBS washes. After being blocked in blocking solution (5% Goat Serum Albumin; Sigma) for 1 h, the sections were then incubated with the primary antibody against MAP2 (1:20, Proteintech) overnight. After two PBS washes and subsequent incubation with secondary antibody (ASS3403; Abgent, San Diego, CA, USA) for 30 min, the sections were then incubated with DAB (Solarbio, DA1010) till proper color appeared.
Goat serum albumin
Manufactured by Merck Group
Sourced in United States
Goat serum albumin is a laboratory product derived from the serum of goats. It is a purified protein that serves as a standard for various biochemical and immunological applications. Goat serum albumin is commonly used in cell culture media, enzyme-linked immunosorbent assays (ELISA), and other research procedures that require a consistent protein source.
Automatically generated - may contain errors
Lab products found in correlation
Da1010, by Solarbio (1 mentions)
Ass3403, by Abcepta (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
Fitc labeled goat anti mouse igg, by Abcam (1 mentions)
Dapi staining solution, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal igg1, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cyanine3 (cy3), by Proteintech (1 mentions)
4 protocols using goat serum albumin
1
Immunohistochemical Staining of MAP2
2
Immunocytochemistry of Connexin 43 in hUCSCs
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Cx43-hUCSCs were cultured on glass cover slips in six well plates and grown to 80% confluence. Cells were washed with PBS and fixed with 40 mg/L paraformaldehyde (Sigma, USA) for 25 min, then blocked with 10% goat serum albumin (Sigma, USA) for 20 min before staining. A drop of DAPI staining solution (Lifetechnologies, USA, 1 mg/ml) was added to stain the cells for 5 min. The slips of cells were washed with PBS, and a drop of 1:100 diluted mouse anti-human Cx43 monoclonal antibody (Abcam, USA) was added and incubated for 24 h at 4°C. Samples were warmed to room temperature for 1 h, washed with PBS, 1 drop of 1:100 diluted FITC-labeled goat anti-mouse IgG (Abcam, USA) was added and incubated for 1 h at room temperature. Mouse monoclonal IgG1 (Abcam, USA) was used as isotype control as instructed by manufactures. After washing with PBS, the slips were sealed using 60% buffering glycerol (Sigma, USA) and imaged by a laser confocal microscope (Leica, Germany) with a fast scanning mode.
3
Drosophila Ovary Immunostaining Protocol
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Ovaries from 3–7-day-old female offspring of dysgenic crosses were dissected and immediately fixed with 4% EM-grade methanol-free paraformaldehyde (Thermo Scientific). Ovaries were washed with 0.1% Triton X-100 in PBS and blocked with 5% goat serum albumin (Sigma-Aldrich). Primary antibody concentrations were as follows: anti-Hts 1B1 1:4 (DSHB [79 (link)]), anti-Vasa 1:40 (DSHB), anti-Bruno 1:1,000 (provided by Paul MacDonald [50 (link)]), and anti-Orb 4H8 and 6H4 1:20 (DSHB [50 (link)]). Secondary antibody concentrations were 1:500.
4
Immunofluorescence Staining of OECs
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The OEC were rinsed three times in phosphate-buffered saline (PBS) before being fixed in 4% PFA for 20 min, and then the cells were rinsed another three times in PBS. For immunofluorescence, the cells were pre-incubated with 10% goat serum albumin (Sigma) for 60 min at room temperature and rinsed three times in PBS, followed by a 12 h incubation at 4 °C with a rabbit anti-rat P75NTR monoclonal antibody (1:50, Proteintech, Chicago, IL, USA). The secondary goat anti-rabbit antibodies Cy3 (Proteintech) were used at 1:500, and the cells were incubated for 60 min at room temperature. At the 55th min, DAPI (100 ng/mL) was added. Then, the cells were rinsed three times in PBS before being mounted in 50% glycerol. Under 200× magnification, 5 different microscopic fields (200×) were randomly chosen. The number of OEC in each field was calculated and summed to obtain an average number of OEC in each group.
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