Cytospin-slides were fixed in 100% acetone at room temperature for 15 min. For the collagen-1 staining, slides were pre-incubated with 10% normal goat serum (Sigma, G9023) in block buffer (1% Blocking Reagent, Roche, in PBS according to the manufacturer’s protocol) for 30 min. After rinsing with PBS, slides were incubated for 60 min with mouse anti-human collagen-1 (Abcam, ab6308, 1:2000) or Isotype control in block buffer. As second (goat anti-mouse antibody biotin-labeled) and third antibody (streptavidin, alkaline phosphatase (AP) conjugate) we used the Link-Label kit from Biogenex (link: HK-325-UM, label HK321-UK) according to the manufacturer’s protocol. To detect the collagen-1 positive cells we used New Fuchsin Alkaline Phosphatase Substrate Solution (0.01% New Fuchsin, 0.02% Sodium Nitrite, 0.03% Naphthol AS-BI Phosphate, 1 mM Levamisole, in 0.2 M Tris-HCl, pH 8.5). Cells were counterstained with hematoxylin (Sigma, Gill No. 3), dried and mounted in Vecta Mount (Vector, Burlingame, CA, USA). For CD15 detection we used the same protocol, but with different antibodies; slides were pre-incubated with normal rabbit serum (Sigma, R9133) and subsequently stained with mouse anti-human CD15-FITC (BD Biosciences, HI98) or isotype control and rat anti-FITC AP conjugate (Sigma, A4843).
R9133
Manufactured by Merck Group
R9133 is a laboratory equipment product. It is a multipurpose device designed for various applications in scientific research and analysis. The core function of R9133 is to provide a controlled environment for conducting experiments and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Merck Group (1 mentions)
Blocking reagent, by Roche (1 mentions)
Ab6308, by Abcam (1 mentions)
G9023, by Merck Group (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Axiocam mrc5, by Zeiss (1 mentions)
Am2616, by Thermo Fisher Scientific (1 mentions)
Ab64211, by Abcam (1 mentions)
Bloxall, by Vector Laboratories (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Tug905, by Merck Group (1 mentions)
Gw9508, by Bio-Techne (1 mentions)
Sc 546, by Santa Cruz Biotechnology (1 mentions)
4 protocols using r9133
1
Collagen-1 and CD15 Immunostaining Protocol
2
Immunohistochemical Analysis of Oxytocin and Related Proteins
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Samples were fixed and stained as described in Rapacz-Leonard55 (link), except for the use of 0.5% BSA (AM2616, Thermo Fisher Scientific) for blocking non-specific binding. Briefly, Bloxall (SP-6000, Vector Laboratories) was used for blocking endogenous peroxidase, then 0.5% BSA and slides were incubated overnight with primary antibodies (Table 3 : anti-OXT, anti-OXTR, anti-PTGS2). For detection, an ImmPRESS HRP Universal Antibody (Anti-Mouse IgG/Anti-Rabbit IgG, Peroxidase) Polymer Detection Kit (MP-7500, Vector Laboratories) for rabbit antibody and an ImmPRESS HRP Anti-Goat IgG (Peroxidase) Polymer Detection Kit (MP-7405, Vector Laboratories) for goat antibodies were used. Slides were photographed using a Zeiss microscope (Axio Imager.M2, Zeiss) with an AxioCam MRC 5 (Zeiss).
As positive controls, equine corpus luteum and the endometrium from a non-pregnant mare in late luteal phase were used. For negative controls, blocking peptides were used. Omission controls were performed with only antibody diluent (ab64211, Abcam). Replacement controls were performed with normal serum instead of primary antibody, using either rabbit serum (R9133, Sigma Aldrich) or goat serum (PK-4010, Vector Laboratories). No isotype control was performed because of the polyclonality of the antibodies.
As positive controls, equine corpus luteum and the endometrium from a non-pregnant mare in late luteal phase were used. For negative controls, blocking peptides were used. Omission controls were performed with only antibody diluent (ab64211, Abcam). Replacement controls were performed with normal serum instead of primary antibody, using either rabbit serum (R9133, Sigma Aldrich) or goat serum (PK-4010, Vector Laboratories). No isotype control was performed because of the polyclonality of the antibodies.
3
Placental Immunohistochemical Analysis
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Placenta tissue specimens were fixed in 10% neutral buffered formalin for at least 1 day and embedded in paraffin, after which 5-μm sections were placed on coated glass slides designed for immunohistochemistry (IHC) processing. Hematoxylin and eosin-stained slides were also produced for histological examination of each placenta. Normal rabbit serum (R9133, used at 1 μg IgG/mL; Sigma–Aldrich) was used as a negative control. Placenta tissue specimens from 11 NW women and 6 OB women were blindly screened with anti-insulin receptor antibody, and subsequently, one representative from each group was selected for performing additional markers. Antibodies and concentrations used are listed in Supplementary Table 3 . Serial sections (5 microns each) were immunohistochemically stained using EnVision+ horseradish peroxidase Rabbit DAB+ (K4011; Dako Agilent, Santa Clara, CA) to mark the sites of antibody binding with a brown deposit as previously described by Kliman et al. [33 (link)]. Antibody concentrations were chosen to produce strong staining in the positive cellular structures without background staining.
4
GPR40 Agonists Regulate Neurogenesis
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For central administration of GPR40 exogenous ligands, 7-week old mice were submitted to cannula implantation in the right lateral ventricle under xylazine (10 mg/kg, ip) and ketamine (100 mg/kg, ip) anesthesia. The coordinates were as follows: anteroposterior, 0.34 mm; lateral, 1.0 mm; and depth, 2.2 mm. The efficiency of cannula placement and viability was confirmed by icv administration of angiotensin II and measurement of the drinking response. Ventricular-cannulated mice were treated daily for 7 days with 2.0 μL of vehicle [1:1:3; Ethanol/ DMSO/ artificial cerebrospinal fluid (Tocris)], GW9508 (2.0 mM; Tocris Bioscience) or TUG905 (2.0 mM, synthesized as previously described50 (link).
BrdU (Sigma) was used to evaluate cell proliferation and survival. BrdU is a thymidine analogue that is incorporated into the DNA double-helix during the S-phase of the cell cycle, and thus marks actively proliferating cells70 (link). All animals received BrdU (0.1 M phosphate-buffered saline [PBS], pH = 7.2; 10 μg/day icv and 50 mg 1 × day ip) and were euthanized 1 or 28 days later (Fig.1 A) to assay proliferation or survival of new cells, respectively.
For some experiments, mice were treated either with an immunoneutralizing rabbit anti-BDNF antibody (0.8 μg/100 μL, sc-546, Santa Cruz Biotechnology) or a pre-immune rabbit serum (R9133, Sigma), by ip injection every 3 days for 2 weeks.
BrdU (Sigma) was used to evaluate cell proliferation and survival. BrdU is a thymidine analogue that is incorporated into the DNA double-helix during the S-phase of the cell cycle, and thus marks actively proliferating cells70 (link). All animals received BrdU (0.1 M phosphate-buffered saline [PBS], pH = 7.2; 10 μg/day icv and 50 mg 1 × day ip) and were euthanized 1 or 28 days later (Fig.
For some experiments, mice were treated either with an immunoneutralizing rabbit anti-BDNF antibody (0.8 μg/100 μL, sc-546, Santa Cruz Biotechnology) or a pre-immune rabbit serum (R9133, Sigma), by ip injection every 3 days for 2 weeks.
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