Free-floating basal forebrain tissue samples (every 6th section; approximately Bregma: 1.60 mm– 0.20 mm based on the atlas of Paxinos and Watson [41 ]) were washed in 0.1 M PBS, incubated in 0.6% H2O2 to inhibit endogenous peroxidases, and blocked with normal serum (MP Biomedicals, Solon, OH). Sections were incubated in a primary antibody solution containing blocking solution and either goat anti-choline acetyltransferase (ChAT; Millipore, Temecula, CA, Cat. #AB144P), rabbit anti-TrkA (Millipore, Cat. #06–574), mouse anti-p75NTR (Millipore, Cat. #MAB365), or rabbit anti-phosphorylated NF-κB p65 Ser 536 (pNF-κB p65; Abcam, Cambridge, MA, Cat. #ab86299) for 24 hr at 4°C. Sections were washed with PBS, incubated for one hr in a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and incubated for one hr in avidin-biotin complex solution (Vectastain ABC Kit; Vector Laboratories). The chromogen, nickel-enhanced diaminobenzidine (Sigma-Aldrich), was used to visualize immunoreactivity. Tissue was mounted onto slides, dehydrated, and cover slipped. Negative control for non-specific binding was conducted on separate sections employing the abovementioned procedures omitting the primary antibody.
Normal serum
Manufactured by MP Biomedicals
Sourced in United States
Normal serum is a laboratory reagent used as a control substance in various biochemical and immunological analyses. It serves as a reference standard to validate the accuracy and reliability of test results. Normal serum is typically derived from pooled human or animal sources and undergoes rigorous quality control measures to ensure consistent composition and performance.
Automatically generated - may contain errors
Lab products found in correlation
Vectastain abc kit, by Vector Laboratories (5 mentions)
Biotinylated secondary antibody, by Vector Laboratories (4 mentions)
Nickel enhanced diaminobenzidine, by Merck Group (4 mentions)
P nf κb p65, by Abcam (1 mentions)
Rabbit anti trka, by Merck Group (1 mentions)
Mouse anti p75ntr, by Merck Group (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Goat anti chat, by Merck Group (1 mentions)
Mouse anti tlr4, by Abcam (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
Goat anti doublecortin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nestin, by Merck Group (1 mentions)
5 protocols using normal serum
1
Basal Forebrain Tissue Immunohistochemistry
2
Immunohistochemical Staining of Basal Forebrain
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Free-floating basal forebrain tissue was washed in 0.1 M PBS, incubated in 0.6% H2O2 to inhibit endogenous peroxidases, and blocked with normal serum (MP Biomedicals, Solon, OH, United States). Sections were incubated in a primary antibody solution containing blocking solution and goat anti-ChAT (Millipore, Temecula, CA, United States, Cat. #AB144P, RRID:AB_2079751 ), mouse anti-TLR4 (Abcam, Cat. #ab22048, RRID:AB_446735 ), or mouse anti-NeuN (Millipore, Cat. #MAB377, RRID:AB_2298772 ) for 24 h at 4°C. Sections were washed with PBS, incubated for 1 h in a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, United States), and incubated for 1 h in ABC solution (Vectastain ABC Kit; Vector Laboratories). The chromogen nickel-enhanced DAB (Sigma-Aldrich) was used to visualize immunoreactivity. Tissue was mounted onto slides, dehydrated, and cover slipped. Negative controls for non-specific binding were conducted on separate sections employing the above-mentioned procedures, omitting the primary antibody.
3
Immunohistochemical Staining of Microglia
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Free-floating sections were washed in 0.1 M PBS, incubated in 0.3% H2O2 for 30 min, washed again in PBS, and blocked for 1 h at room temperature in 0.25% Triton-X100/5% normal serum (MP Biomedicals, Solon, OH, Cat. # 19135680). Sections were transferred directly from the block to primary antibody (rabbit anti-Iba1, WAKO, Japan) diluted in blocking solution and were incubated overnight at 4 °C. Sections were then washed in PBS, incubated for 1 h in biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA), and washed and incubated for 1 h in avidin–biotin complex solution (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, Cat. # PK6100). The chromogen, nickel-enhanced diaminobenzidine (Sigma-Aldrich, St. Louis, MO, Cat. # D5637), was used to visualize immunoreactivity. Tissue was mounted onto slides, dehydrated, and coverslipped.
4
Immunohistochemical Staining of Hippocampal Markers
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Free-floating hippocampal sections (every 12th section) were washed in 0.1 M PBS, incubated in 0.3% H2O2 to inhibit endogenous peroxidases, and blocked with the appropriate normal serum (MP Biomedicals, Solon, OH). Sections were incubated in a primary antibody solution containing blocking solution, and either goat anti-doublecortin (Santa Cruz Biotechnology, Santa Cruz, CA; Cat. #sc-8066), rabbit anti-Ki-67 (Abcam, Cambridge, MA; Cat. #ab66155), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, Danvers, MA; Cat. #9661), rabbit anti-phosphorylated NF-κB p65 Ser 276 (pNF-κB p65; Santa Cruz Biotechnology; Cat. #sc-101749), or mouse anti-nestin (Millipore, Temecula, CA; Cat. #MAB5326) for 24 hr at 4°C. Sections were then washed with PBS, incubated for 1 h in the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and incubated for 1 h in avidin-biotin complex solution (Vectastain ABC Kit; Vector Laboratories). The chromogen, nickel-enhanced diaminobenzidine (Sigma-Aldrich, St. Louis, MO), was used to visualize immunoreactivity. Tissue was mounted onto slides, dehydrated, and cover slipped. Negative control for non-specific binding was conducted on separate sections employing the abovementioned procedures omitting the primary antibody.
5
Immunohistochemical Analysis of Basal Forebrain
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Free-floating basal forebrain tissue samples (every 6th section; approximate Bregma: 1.60–0.20 mm based on the atlas of Paxinos and Watson, 1998 ; see Figure 1C ) were washed in 0.1 M PBS, incubated in 0.6% H2O2 to inhibit endogenous peroxidases, and blocked with normal serum (MP Biomedicals, Solon, OH). Sections were then incubated in a primary antibody solution for 24 h at 4°C. Primary antibodies, dilutions, and validation information are included in Table 1 . Sections were washed with PBS, incubated for 1 h in a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and incubated for 1 h in avidin-biotin complex solution (Vectastain ABC Kit; Vector Laboratories). The chromogen, nickel-enhanced diaminobenzidine (Sigma-Aldrich), was used to visualize immunoreactivity. Tissue was mounted onto slides, dehydrated, and cover slipped. Negative control for non-specific binding was conducted on separate sections employing the abovementioned procedures omitting the primary antibody.
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