Sections from the mPFC (3 sections spaced at 120μm intervals per animal) were taken out of cryoprotectant and rinsed three times (five min each) in Tris-buffered saline (TBS), pH 7.6. They were then placed in blocking solution consisting of 1% hydrogen peroxide, 20% normal goat serum (NGS), and 1% bovine serum albumin (BSA) in TBS for 30 min. They were then incubated in an anti-TH primary antibody (Mouse monoclonal, Millipore; MAB318) diluted at 1:1000 with 2% NGS and 0.3% triton-x-100 in TBS (TTG) for 48 hours. Sections were then rinsed in TTG three times for five minutes, followed by incubation in 5μg/ml biotinylated goat, anti-mouse secondary antibody (Vector; BA-9200) in TTG for 90 min. Sections were then rinsed twice in TTG and twice in TBS (5 min each) before being placed into avidin-biotin-complex diluted in TBS (Vector; PK-6100) for one hour at room temperature. Lastly, sections were rinsed three times in TBS, then reacted with Diaminobenzidin (DAB) (Sigma fast tabs; D4418). After excess DAB was rinsed, sections were mounted on gelatin-coated slides and cover-slipped with Permount for analysis.
D4418
Manufactured by Merck Group
Sourced in United States
D4418 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis applications. The core function of D4418 is to provide a controlled environment for various experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Mab318, by Merck Group (1 mentions)
Ba 9200, by Vector Laboratories (1 mentions)
Pk 6100, by Vector Laboratories (1 mentions)
Dt 1bon1000 96, by Immunodiagnostic Systems (1 mentions)
Wga lectin, by Merck Group (1 mentions)
Crosslaps for culture ctx 1 elisa kit, by Immunodiagnostic Systems (1 mentions)
Sc 32790l, by Santa Cruz Biotechnology (1 mentions)
Ma1 27198, by Thermo Fisher Scientific (1 mentions)
Rankl, by R&D Systems (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Vectastain elite abc system, by Vector Laboratories (1 mentions)
Discovery v12 microscope, by Zeiss (1 mentions)
3 30 diaminobenzidine, by Merck Group (1 mentions)
6 protocols using d4418
1
Immunohistochemical Labeling of Tyrosine Hydroxylase
2
Quantifying Bone Resorption Using Osteoclasts
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For bone resorption assays, BMDMs or pre-osteoclasts were seeded and cultured on bovine cortical bone slices (DT-1BON1000-96; Immunodiagnostic Systems) with 20 ng/ml M-CSF and 30 ng/ml RANKL (R&D Systems; Wu et al., 2017 (link); Zhang et al., 2018 (link); Zhu et al., 2020 (link)), in the presence or absence of galectin-3 (8259-GA; R&D Systems), galectin-3C (10110-GA; R&D Systems), GCS-100 (La Jolla Pharmaceutical), RAP (4480-LR; R&D Systems), an anti–galectin-3 blocking antibody (sc-32790L; Santa Cruz), or an anti-Lrp1 blocking antibody (MA1-27198; Thermo Fisher Scientific; Chen et al., 2015 (link); Demotte et al., 2010 (link); John et al., 2003 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). After the indicated culture period, bone samples were sonicated in PBS, stained with 20 μg/ml WGA-lectin (L3892; Sigma-Aldrich) for 45 min and then incubated with DAB tablets (D4418; Sigma-Aldrich) for 15 min. Image J software was used to quantify the resorbed area. The concentration of the CTX-I was measured using the CrossLaps for Culture CTX-I ELISA kit (AC-07F1; Immunodiagnostic Systems) according to the manufacturer’s instructions.
3
Phospho-Tau Immunohistochemistry Protocol
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Sections were rinsed in tris-buffered saline (TBS), treated with 0.6% hydrogen peroxide in TBS containing 0.1% Triton-X (TBST) for 20 min, and washed again before undergoing antigen retrieval in 10 mM sodium citrate pH 6.0 for 30 min at 80°C. Non-specific binding was blocked by 90 min incubation in TBST containing 5% normal goat serum, before sections were transferred to primary antibody diluted 1:500 in blocking solution for overnight incubation at 4°C (mouse anti-phospho serine 202/threonine 205 tau antibody (AT8, Life Technologies, MN1020) or conformation specific mouse anti-tau (MC1, made by Dr. Peter Davies). Sections were rinsed in TBS before being incubated in biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, BA-1000) diluted 1:500 in blocking solution. After several rinses in TBS, tissue was incubated for 30 min at room temperature with HRP-avidin conjugate diluted 1:50 in TBS. Sections were developed with DAB (D4418, Sigma), then mounted, dehydrated, and coverslipped with Permount.
4
Immunohistochemical Analysis of SCR and UBX
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For each genotype, at least 15 third instar wandering larvae were dissected and fixed in 3.7% paraformaldehyde for 20 min at room temperature, then immunostained according to [82 (link),83 (link)] using rat polyclonal anti-SCR antibody (1:100 [82 (link)]) or mouse anti-UBX monoclonal antibody (1:20; FP3.38 [84 (link)]). The universal biotinylated antibody (Vector Laboratories, CA, USA) was used at a 1:200 dilution. Staining was performed with VECTASTAIN Elite ABC system (Vector Laboratories, CA, USA) using DAB as substrate (D4418, Sigma, St. Louis, MO, USA). Note that for a given antibody, discs of all genotypes were incubated for the same length of time in DAB. Imaginal discs were mounted in PBS:glycerol (50:50). All pictures were acquired with a QICAM Fast 1394 digital camera, at × 100 magnification. Staining was quantified by calculating the percentage of stained area in the discs using Image J. SCR positive area was measured as a percentage of the total leg disc area, and UBX positive area was evaluated relative to the presumptive wing blade and hinge area of the wing disc. Statistical significance of results was evaluated using t-test.
5
Zebrafish Proliferation Assay via PH3 Staining
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Zebrafish proliferation was measured by anti-phospho-Histone H3 (PH3) staining. Briefly, chorions of 24 hpf zebrafish embryos were removed using gentle forceps and fixed with 4% PFA overnight at 4°C. Embryos were then washed twice in PBS and incubated for 7 min in −20°C acetone. Acetone was removed and embryos were washed in double-distilled water followed by two washes in PBST (0.1% Tween in PBS) for 5 min. Embryos were blocked for 2 h at room temperature in blocking solution (10% FCS, 1% DMSO in PBST). After blocking, embryos were incubated in blocking solution with a 1:750 dilution of rabbit p-Histone H3 antibody (Cell Signaling Technology; #9701) overnight at 4°C. Post antibody incubation, embryos were washed 4 × 5 min in PBST and incubated for 2 h at room temperature with a 1:300 dilution of donkey anti-rabbit HRP conjugated antibody (Abcam; ab97085). Embryos were washed 4 × 15 min in PBST and incubated for 10 min in 3,3′-diaminobenzidine (DAB) solution (D4418; Sigma-Aldrich). After incubation, embryos were washed three times in PBST. Images were captured by Discovery.V12 microscope (Zeiss) and analyzed for proliferation capacity by measuring pixel integrated density of zebrafish heads using ImageJ software.
6
Neuroanatomical and Molecular Analysis of ABA
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At the end of the 12 h light phase on day 6 of the ABA induction phase, rats were deeply anesthetized (chloral hydrate, 500 mg/kg, i.p., 2 mL/kg) and perfused transcardially in ice-cold 0.1 M PBS (pH 7.4), followed by 4% paraformaldehyde. Thereafter, their brains were post-fixed overnight in 4% paraformaldehyde and stored in 0.1% NaN3-PBS at 4 °C. Free-floating coronal brain sections of 40 μm thickness were vibratome-cut at the level of the prelimbic and infralimbic prefrontal cortex (Bregma: +3.72 to +2.52), nucleus accumbens shell and core (Bregma: +2.28 to +1.08), dorsal hippocampus (Bregma: −2.16 to −4.08), and ventral hippocampus (Bregma: −5.16 to −6.12), according to the atlas of Paxinos and Watson [19 ]. For Arc and c-Fos quantification, sections were pre-incubated in BSA/normal donkey serum blocking solution and then immunoreacted with mouse monoclonal Arc (C-7) (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-17839) and sheep polyclonal c-Fos (1:2000, Sigma Aldrich, St Louis, MO, USA; AB1584) primary antibodies. The reaction was then amplified using the proper biotinylated secondary antibody and visualized by the classic avidin–peroxidase complex (ABC, Vector Laboratories, Burlingame, CA USA; VEC.PK-6100) protocol, using 3,30-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA; D4418) as a chromogen.
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