Brain sections were immunostained for somatostatin (Santa Cruz Biotechnology, Santa Cruz, CA; catalog number SC7819) as described in Spiegel et al. (2013) (link). Briefly, free-floating sections were washed in 0.1 M phosphate-buffered saline (PBS), and then endogenous peroxidases were quenched in 0.3% hydrogen peroxide in PBS. After additional PBS washes, sections were blocked in 5% normal horse serum in PBS with 0.3% Triton. Sections were then incubated with primary antibody at a dilution of 1:1,600 in PBS containing 0.15% Triton and 3% normal serum for 72 hr at 4°C with agitation. Following primary antibody incubation, sections were washed in PBS and reacted with biotinylated secondary antibody horse antigoat IgG (Vector Laboratories, Burlingame, CA) diluted in PBS with 0.15% Triton and 5% normal horse serum for 45 min. The secondary antibody was detected with avidin-biotin complex (ABC Elite; Vector Laboratories) and the avidin-biotin complex was visualized with nickel-enhanced diaminobenzadine (Vector Laboratories). Tissue sections were mounted onto gelatin coated slides and dried, dehydrated with increasing ethanol concentrations, cleared in xylene, and coverslipped using DPX mounting media.
Horse anti goat igg
Manufactured by Vector Laboratories
Horse anti-goat IgG is a secondary antibody produced in horses and directed against goat immunoglobulin G (IgG). It is used as a detection reagent in various immunological techniques, such as ELISA, Western blotting, and immunohistochemistry, to identify the presence of goat IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Diaminobenzidine, by Merck Group (2 mentions)
Sc 271619, by Santa Cruz Biotechnology (2 mentions)
Streptavidin conjugated with horse radish peroxidase, by Zymo Research (2 mentions)
Somatostatin, by Santa Cruz Biotechnology (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Miniprotein 3 wet transfer unit, by Bio-Rad (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Srp0292, by Merck Group (1 mentions)
Af534, by R&D Systems (1 mentions)
Anti cathepsin s, by Santa Cruz Biotechnology (1 mentions)
Biotinylated wfa lectin, by Vector Laboratories (1 mentions)
6 protocols using horse anti goat igg
1
Immunostaining of Brain Sections for Somatostatin
2
Western Blot Analysis of Hippocampal Proteins
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Hippocampal tissues were homogenized and centrifuged at 4?, 12,000 rpm for 15 min. The protein samples were transferred onto PVDF membranes using a Bio-Rad miniprotein-III wet transfer unit, then blocked with 5% skimmed milk dissolved in TBST at room temperature for 1 h. After washing with TBST buffer three times, membranes were incubated at 4? overnight with one of the primary antibodies listed in Table 1 . Peroxidase-conjugated goat anti-rabbit IgG, horse anti-rat IgG or horse anti-goat IgG (1:2000; all from Vector Laboratories) was used, and bands were visualized using ECL plus detection system. GADPH was utilized as an internal control for protein loading and transfer efficiency.
3
Tissue Fixation and Immunohistochemistry for uPAR
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Tissues were fixed overnight in 10% formalin, embedded in paraffin and cut into 5-μm sections. Sections were subjected to H&E staining. Immunohistochemical staining was performed following standard protocols. The following antibodies were used: anti-mouse uPAR (AF534, R&D, DCL0521042; 1:50 dilution) and horse anti-goat IgG (30116; Vector Laboratories, ZH0526). Three fields per section were counted per sample with Fiji-ImageJ and averaged to quantify the percentage of uPAR-positive area per field. SA-β-gal staining was performed as previously described47 (link) at a pH of 5.5 for mouse tissues. Specifically, fresh frozen tissue sections were fixed with 0.5% glutaraldehyde in PBS for 15 min, washed with PBS supplemented with 1 mM MgCl2 and stained for 5–8 h in PBS containing 1 mM MgCl2, 1 mg ml−1 X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide. Tissue sections were counterstained with eosin. Three fields per section were counted with ImageJ and averaged to quantify the percentage of SA-β-gal-positive area per field.
4
Immunohistochemical Detection of Cathepsin-S
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Free-floating tissue sections were carried through antigen retrieval in citric acid buffer (0.1 m citric acid and 0.2 m Na2HPO4), heated to 80°C for 30 min, and incubated in biotinylated WFA lectin (catalog #B-1355, Vector Labs) or the mouse monoclonal primary antibody anti-cathepsin-S (1:500, sc-271619, Santa Cruz Biotechnology Inc.) for 48 h, and subsequently in biotinylated secondary antibody (horse anti-goat IgG; 1:500; Vector Labs), followed by streptavidin conjugated with horse-radish peroxidase for 2 h (1:5000 μl, Zymed), and, finally, in nickel-enhanced diaminobenzidine/peroxidase reaction (0.02% diaminobenzidine, Sigma-Aldrich, 0.08% nickel-sulfate, 0.006% hydrogen peroxide in PB). All solutions were made in PBS with 0.2% Triton X-100 (PBS-Tx) unless otherwise specified. Immunostained sections were mounted on gelatin-coated glass slides, coverslipped, and coded for blinded quantitative analysis. All sections included in the study were processed simultaneously within the same session to avoid procedural differences. Omission of the primary or secondary antibodies did not result in detectable signal, and preabsorption of mouse anti cathepsin-S with 300 nanograms of active human cathepsin-S (SRP0292, Sigma-Aldrich) did not result in detectable immunolabeling signal.
5
Immunohistochemical and Senescence Analysis
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Tissues were fixed overnight in 10% formalin, embedded in paraffin and cut into 5-μm sections. Sections were subjected to hematoxylin and eosin (H&E) staining. Immunohistochemical staining was performed following standard protocols. The following antibodies were used: anti-mouse uPAR (R&D, AF534, lot DCL0521042, 1:50), Horse anti-goat IgG (Vector laboratories, 30116; lot ZH0526). Three fields per section were counted per sample with ImageJ and averaged to quantify the percentage of uPAR+ area per field. SA-β-gal staining was performed as previously described47 (link) at pH 5.5 for mouse tissues. Specifically, fresh frozen tissue sections were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 min, washed with PBS supplemented with 1 mM MgCl2 and stained for 5–8 h in PBS containing 1 mM MgCl2, 1 mg ml−1 X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide. Tissue sections were counterstained with eosin. Three fields per section were counted with ImageJ and averaged to quantify the percentage of SA-β-gal+ area per field.
6
Immunohistochemical Analysis of Cathepsin-S
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Free-floating tissue sections were carried through antigen retrieval in citric acid buffer (0.1 M citric acid, 0.2 M Na2HPO4) heated to 80 degrees °C for 30 minutes, and incubated in biotinylated WFA lectin (cat#B-1355, Vector Labs) or the mouse monoclonal primary antibody anti-cathepsin-S (1:500, sc-271619, Santa Cruz Biotechnology Inc.) for 48 hours, and subsequently in biotinylated secondary antibody (horse anti-goat IgG; 1:500; Vector Labs, Inc. Burlingame, CA), followed by streptavidin conjugated with horse-radish peroxidase for two hours (1:5000 µl, Zymed, San Francisco, CA), and, finally, in nickel-enhanced diaminobenzidine/ peroxidase reaction (0.02% diaminobenzidine, Sigma-Aldrich, 0.08% nickel-sulphate, 0.006% hydrogen peroxide in PB). All solutions were made in PBS with 0.2% Triton X (PBS-Tx) unless otherwise specified. Immunostained sections were mounted on gelatin-coated glass slides, coverslipped and coded for blinded quantitative analysis. All sections included in the study were processed simultaneously within the same session to avoid procedural differences. Omission of the primary or secondary antibodies did not result in detectable signal, and pre-absorption of mouse anti cathepsin-S with 300 nanograms of active human cathepsin-S (SRP0292, Sigma-Aldrich, St. Louis, MO) did not result in detectable immunolabeling signal.
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