All procedures were performed as previously described [43 (link), 44 ]. Briefly, mice were anesthetized by pentobarbital sodium and perfused with saline. Brains were fixed with 4% paraformaldehyde (w/v) for 7 days. Fixed brains were transferred to 30% sucrose solution for 3 days. Coronal sections were cut throughout the whole brain. In immunohistochemistry (IHC), brain slices were blocked with 10% goat serum (Abcom, ab7481) in PBS containing 0.2% Triton X-100 (Sigma, V900502), and incubated with Iba1 primary antibody (WAKO, 019-19741, 1:400), biotinylated goat anti-rabbit IgG, and streptavidin-conjugated horseradish peroxidase using VECTASTAIN® ABC-HRP kit (Vector Laboratories, PK-4000). Iba1 stains were visualized with 3,3′-diaminobenzidine (Sigma-Aldrich), scanned and analyzed by stereo investigator (MicroBrightfield). In immunofluorescence, brain slices were blocked, and incubated with primary and secondary antibodies as follow: Iba1 (Novus Biologicals, NB100-1028, 1:400), ASC (Cell Signaling Technology, D2W8U, 1:500), Alexa Fluor 546-conjugated (Invitrogen, A-11056, 1:400), and FITC-conjugated (Abcam, ab6798, 1:400). Hoechst 33258 (Sigma) were incubated for the visualization of nuclear morphology.
Nb100 1028
Manufactured by Cell Signaling Technology
NB100-1028 is a primary antibody product from Cell Signaling Technology. It is designed to detect endogenous levels of total p38 MAPK protein.
Automatically generated - may contain errors
Lab products found in correlation
Iba1 primary antibody, by Fujifilm (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ab6798, by Abcam (1 mentions)
Ab7481, by Merck Group (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
A11056, by Abcam (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Anti neun, by Merck Group (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A10040, by Thermo Fisher Scientific (1 mentions)
Anti cd68, by Bio-Rad (1 mentions)
Anti brdu, by Bio-Rad (1 mentions)
Anti ki67, by Abcam (1 mentions)
B2883, by Merck Group (1 mentions)
Asp175, by Cell Signaling Technology (1 mentions)
2 protocols using nb100 1028
1
Immunohistochemical and Immunofluorescence Analysis of Microglia
2
Immunohistochemical analysis of neurogenesis
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BrdU was from Sigma-Aldrich. Antibodies used were as follows: anti-mouse Axl (R&D AF854), anti-Mer (eBioscience DS5MMER and R&D AF591), anti-mouse Gas6 (R&D AF986), anti-BrdU (BU1/75 [ICR1], AbD serotec), anti-Iba1 (Wako 019-19741, 1:200, and Novus NB100-1028), anti-cleaved Casp3 (Asp175, Cell Signaling 9661), anti-doublecortin (DCX, Millipore ab2253), anti-ZnT3 (Sysy 197 002), anti-PSD-95 (Abcam ab18258), anti-CD68 (BioRad MCA1957), anti-NeuN (Chemicon MAB377), anti-Arc (Sysy #156003), anti-Ki-67 (Abcam ab15580), and anti-Ki-67 APC-conjugated (Invitrogen, clone SolA15). Secondary antibodies for immunohistochemistry were fluorophore-conjugated anti-rat (712- 545-153 and 712-165-153 from Jackson ImmunoResearch), anti-goat (A-11056 from Life Technologies, or 705-166-147 from Jackson ImmunoResearch), anti-guinea pig (Molecular Probe A-11073, and Jackson ImmunoResearch 706-165-148 and 706-175-148 were kind gifts from C.Kintner of the Salk Institute, La Jolla, CA, USA), anti-rabbit (A-10040 or A-21206 from Life Technologies and 711-606-152 from Jackson ImmunoResearch), and anti-mouse (A-21202 from Molecular Probe, 715-166-150 and 715-176-150 from Jackson ImmunoResearch). Key reagents and their usage were as follows: ThioS (Acros Organics 213150250), Hoechst 33258 in a 1% solution of bisbenzimide in water (Sigma-Aldrich B-2883), and paraformaldehyde (PFA) (Sigma P6148).
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