The selevtive 5-HT2 receptor antagonist ketanserin and the antihistamine cyproheptadine with anti-5-HT properties were purchased from Biomol Research Labs, Inc. Stock solutions were prepared by dissolving each in a 50% DMSO/PBS buffer and diluted further in PBS before injection into mice. The doses of the antagonists (500 nmol per mouse) injected intraperitoneally (i.p.) immediately before PUVA exposure were based on previous studies (18 (link), 28 (link)), in which such concentrations totally blocked UVB-induced immune suppression. 5-HT was purchased from Sigma-Aldrich (product no. H9523, Saint Louis, MO) for i.p. injection (500 nmol per mouse). Anti-5-HT antiserum (produced in rabbit) (Sigma product number S 5545) (100 μg i.p. per mouse) was used for in vivo i.p. injection to neutralize 5-HT activity. As a control, an equal amount of normal rabbit serum (Sigma-Aldrich) was used. Anti-cis urocanic acid antibody was a gift from Mary Norval and a dose of 5 μg i.p. per mouse or an equivalent amount of isotype antibody was injected, as previously described (18 (link), 28 (link)).
Normal rabbit serum
Manufactured by Merck Group
Sourced in United States
Normal rabbit serum is a biological product derived from the blood of healthy rabbits. It contains a natural mixture of proteins, antibodies, and other components found in the serum of rabbits. This serum can be used as a reference or control material in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
Protein block, by Abcam (2 mentions)
Rabbit anti mouse cd34 antibody, by Abcam (2 mentions)
Rabbit specific hrp dap detection ihc kit, by Abcam (2 mentions)
Normal mouse serum, by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Facscanto 2, by BD (1 mentions)
Normal human serum, by Merck Group (1 mentions)
Hoechst, by BD (1 mentions)
Hematoxylin solution, by Merck Group (1 mentions)
Extravidin staining kit, by Merck Group (1 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
3 3 diaminobenzidine (dab), by Fujifilm (1 mentions)
Goat antirabbit igg fitc conjugate, by Merck Group (1 mentions)
Confocal microscope, by Bio-Rad (1 mentions)
Rabbit anti cd2ap, by Santa Cruz Biotechnology (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Endoglycosidase hf, by New England Biolabs (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
17 protocols using normal rabbit serum
1
PUVA-Induced Immunosuppression Modulation
2
Quantifying Neovascularization in PEG Hydrogel Transplants
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To analyse the neovascularisation within transplanted PEG hydrogels, paraffin-sectioned slides were stained for mouse endothelial cells. First, sections were deparaffinised with Xylene and rehydrated in 100% ethanol. Slides were incubated in 3% H2O2 for 30 min at room temperature to block any endogenous peroxidase activity. Then, slides were incubated in 10 mmol/l Tris EDTA (pH 9) solution for 20 min at 96 °C and additional 20 min at room temperature for antigen retrieval. Afterwards, non-specific bindings were blocked by the protein block (Abcam, Cambridge, UK) for 1 h followed by overnight incubation at 4 °C with primary antibodies—rabbit anti-mouse CD34 antibody (1:500 dilution; Abcam, catalog#ab81289) dissolved in Tris-buffered saline with 1% Normal Rabbit Serum (Sigma-Aldrich) and 0.1% bovine serum albumin (Fisher Scientific, Waltham, MA, USA). Following incubation overnight, slides were treated for 20 min each with the biotinylated goat anti-polyvalent and streptavidin peroxidase, provided in Rabbit Specific HRP/DAP Detection IHC kit (Abcam). Haematoxylin was used for counterstaining. Healthy mouse uterus was used as positive-control and negative-control slides were incubated without the presence of primary antibody (Supplementary Figure S3 ). The identical procedure was followed for both positive and negative control as described.
3
Flow cytometry analysis of macrophages
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Cells were harvested by scraping and resuspended in FACS staining buffer (0.9% normal mouse serum, 0.9% normal rabbit serum, 0.9% normal human serum [all Sigma-Aldrich], 3% BSA, 2 mM EDTA in PBS). After antibody staining and washing, cells were resuspended in PBS with Hoechst (BD Biosciences). Counting was performed using a FACSCanto-II (BD Biosciences), and data were analyzed with FlowJo software (TreeStar) by gating on F4/80 and CD11b positive cells.
4
Immunohistochemical Analysis of Oviduct Tissue
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The serial sections of the oviduct tissues were incubated with 10% normal goat serum to reduce background staining caused by the second antibody. The sections were then incubated with primary polyclonal antibody (1:500) against leptin (Y-20) (Santa Cruz Biotechno logy, Santa Cruz, CA, USA), leptin receptor (H-300) (Santa Cruz Biotechnology) and PPARγ2 (bs-7114R) (Beijing Biosynthesis Biotechno logy Co., Beijing, China) for 12 h at 4°C. The control sections were treated with normal rabbit serum (Sigma) instead of the primary antisera. The sections were then incubated with a second antibody for 0.5 h at room temperature, goat anti-rabbit lgG conjugated with biotin and peroxidase with avidin, The sections were visualized using a rabbit ExtrAvidin™ staining kit (Sigma) in 150 mL of 0.05 M Tris-HCl buffer, pH 7.6 containing 30 mg 3, 3-diaminobenzidine (Wako, Tokyo, Japan) plus 30 μL H2O2. Finally, the reacted sections for leptin and leptin receptor were counterstained with hematoxylin solution (Merck, Tokyo, Japan).
5
Podocyte Response to LN Patient Plasma
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Podocytes were cultured on sterilised coverslips in medium containing plasma from LN patients or from healthy controls. After 48 h exposure of the podocytes to plasma, the effect on the localisation of α-actinin and CD2AP was determined by immunofluorescent staining. Cells were fixed with paraformaldehyde and permeabilised with triton-X 100. Non-specific binding was blocked with 2%FCS/2%BSA/0.1%Tween/PBS containing 10% normal rabbit serum (sigma), and then cells were incubated with the primary antibodies (rabbit-anti-α-actinin-4 (ImmunoGlobe), rabbit-anti-CD2AP (Santa Cruz Biotechnology)). After washing, goat antirabbit IgG-FITC conjugate (Sigma) was added and the cover slips were mounted on glass slides using Mowiol mountant. The slides were viewed on a Biorad confocal microscope and images captured using LaserSharp 2000 and analysed using confocal assistant V.4.02 software.
6
Immunoblotting and Detection of MR1
7
Osteosarcoma Cell Surface Marker and miR-335 Expression Analysis
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For analysis of cell surface markers, osteosarcoma cells were harvested and resuspended in PBS/0.5% normal rabbit serum (Sigma-Aldrich), and blocked on ice for 15 min. Cells were subsequently labelled with Alexa Fluor® 647 anti-human Stro-1 antibody (BioLegend) and PE anti-human CD117 (c-kit) antibody (BioLegend) for 60 min and maintained on ice until analysis. The expression was assessed by flow cytometry and data were analyzed. The double positive (DP) and double negative (DN) cells were then sorted and collected using a Becton–Dickinson FACSort (San Jose).
In order to divide the cells into different subpopulations according to their miR-335 expression status, we incubated the cells with miR-335-5p Hu-Cy5 SmartFlare™ RNA Detection Probe (Millipore), overnight for 16 h. The fluorescence was then detected and cells were sorted into miR-335-high and miR-335-low subpopulation using a Becton–Dickinson FACSort (San Jose). The collected cells were then send for subsequent experiments.
In order to divide the cells into different subpopulations according to their miR-335 expression status, we incubated the cells with miR-335-5p Hu-Cy5 SmartFlare™ RNA Detection Probe (Millipore), overnight for 16 h. The fluorescence was then detected and cells were sorted into miR-335-high and miR-335-low subpopulation using a Becton–Dickinson FACSort (San Jose). The collected cells were then send for subsequent experiments.
8
Tissue Microarray Immunohistochemistry Protocol
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TMA specimens (cohort 1) and 9 WSIs (of cohort 2) were deparaffinized in xylene and rehydrated through a graded alcohol series. Antigen retrieval step was performed in a water bath at 98°C for 20 minutes in Target Retrieval Solution, pH 9 (Agilent), followed by cooling down in distilled water. To block nonspecific binding, the slide was incubated with PBS containing 0.05% Tween, 0.1% Triton X-100, 3% BSA (Sigma), 5% normal mouse serum (Sigma-Aldrich), 5% normal rabbit serum (Sigma-Aldrich), and 5% normal goat serum (Sigma-Aldrich) for 45 minutes. The slide was then incubated overnight at 4°C with a metal-tagged antibodies panel (Supplementary Table S3) and then washed twice in 0.2% Triton X-100 (Sigma) PBS+/+ for 8 minutes and in PBS+/+ for 8 minutes. Finally, the TMA was incubated with 0.63 μg/mL Cell-ID Intercalator-Ir (Standard Biotools) for 30 minutes at room temperature washed with distilled water and air dried.
9
ChIP Assays for Runt Transcription
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The ChIP assays were performed as described previously (Wang et al., 2007 (link)) using chromatin prepared from 3- to 4-h embryo collections with either a rabbit anti-Runt antibody or normal rabbit serum from Sigma. The primer sequences used in quantitative PCR (qPCR) are available upon request. (Error bars represent the mean SE from three independent immunoprecipitation experiments.)
10
Cryosectioning and Immunofluorescence of Lymph Nodes
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Freshly isolated lymph nodes were fixed in 1% PFA for 1 hr at room temperature, washed with PBS three times, and stored at 4°C in 30% sucrose solution overnight. Fixed samples were then transferred to OCT (Tissue-Tek) and snap-frozen. Thirty µm thick sections were cut via cryostat (Leica). The sections were dried at room temperature for 3 hr. They were blocked using normal rabbit serum (Sigma-Aldrich) in PBS with 0.2% Triton X-100 and then stained with anti-mouse CD3 PE-CF594 (BD) and anti-mouse Bcl6 Alexa Fluor 488 (BD) overnight. After washing, slides were then mounted in fluoromount-G (Southern Biotech) and analyzed via confocal microscopy with Leica SP5 II (Leica Microsystems) using a 25× objective. Images were processed using Imaris and ImageJ. For quantitative analysis of GCs, Bcl6-positive areas in B cell follicles were identified, manually outlined in ImageJ, and their area was then calculated.
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