We analyzed all tissue samples from in vivo experiments. Four μm-thick tissue sections from paraffin-embedded blocks were stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissue sections were incubated in 0.01M citrate buffer (pH 6.0) at 90°C for 60 min, followed by blocking with normal rabbit serum (1:75, Abcam, Cambridge, UK) for 20 min. Subsequently, tissue sections were incubated with anti-F4/80 antibody (1:100, Abcam) and anti-Gr1 antibody (1:200, Abcam) for 2 h. Tissue sections were then incubated with biotinylated anti-rabbit IgE and avidin–biotin horse radish peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 2 h and stained with DAB (Invitrogen, Eugene, OR, USA) for 20 min. Stained tissues were visually inspected using a model Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a CCD camera and computer-assisted image analysis with DP2-BSW (Olympus, Tokyo, Japan). The numbers of inflammatory foci, F4/80+ cells, and Gr1+ cells were counted in two liver sections of each mouse or 10 hepatocytes.
Normal rabbit serum
Manufactured by Abcam
Sourced in United Kingdom
Normal rabbit serum is a biological reagent derived from the blood of healthy rabbits. It contains a natural mixture of proteins, including immunoglobulins, that are present in the serum of rabbits under normal physiological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti f4 80 antibody, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Thermo Fisher Scientific (1 mentions)
Dp2 bsw, by Olympus (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Anti gr1 antibody, by Abcam (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Ab48187, by Abcam (1 mentions)
Ab6640, by Abcam (1 mentions)
Novared, by Vector Laboratories (1 mentions)
Sa 5004, by Vector Laboratories (1 mentions)
Nis elements software, by Nikon (1 mentions)
Anti tslp antibody, by Abcam (1 mentions)
I2 u microscope, by Nikon (1 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Olympus (1 mentions)
4 protocols using normal rabbit serum
1
Quantification of Liver Inflammation
2
Comprehensive Renal Pathology Analysis
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At sacrifice, renal tissue was fixed in 4% paraformaldehyde and subsequently sectioned using a microtome. To evaluate renal pathology, PAS staining was performed to assess protein cast formation, and picrosirius red (PSR) and α-smooth muscle actin staining was used to assess fibrosis. For PSR staining, kidney tissue sections were processed with saturated picric acid solution and then stained with Sirius red F3B (Colour Index 35782). For T-cell staining, rabbit anti-human CD3 antibody (Dakocytomation) was used at a concentration of 0.6 g/L and a dilution of 1:100 in normal goat serum (Vector). For macrophage staining, rat antibody to F4/80 (ab6640, Abcam) was used at a dilution of 1:30 in normal rabbit serum. For CHOP staining, rabbit anti-GADD153 (sc-575, Santa Cruz) was used at 1:40 dilution. To detect IRE1α phosphorylation, anti-IRE1 (phospho S724) antibody (ab48187, Abcam) was used at a dilution of 1:100. Horseradish peroxidase streptavidin (Vector SA-5004) was used in combination with Nova Red (Vector) and hematoxylin (Sigma) to visualize the stained cells.
3
Immunostaining Protocol for AD Lesions
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Immunostaining was as previously described [44 (link)] with minor modifications. Skin tissues obtained from AD lesional mice were subjected to fixation in 4% paraformaldehyde, sectioning, deparaffinization, and rehydration. For immunohistochemistry, tissue slides were incubated in 0.01 M citrate buffer (pH 6.0) at 90 °C for 80 min, followed by blocking with normal rabbit serum (1:75, abcam, Cambridge, UK) for 20 min. Subsequently, slides were incubated with anti-TSLP antibody (1:75; abcam) for 2 h. Sections were then incubated with biotinylated anti-rabbit IgE and avidin-biotin horse radish peroxidase (HRP) complex (abcam) for 2 h and stained with diaminobenzidine (abcam) for 20 min. For immunofluorescence, slides were incubated with anti-IL-4 and anti-CD4 antibodies (1:50 dilution, 5 μg/mL, respectively; eBioscience, San Diego, CA, USA) for 1 h and then incubated with Alexa 488 or Alexa 647 conjugated secondary antibodies (1:200 dilution; abcam) plus 4’,6-diamidino-2-phenylindole (DAPI) for 1 h. All sections were analyzed using a Nikon i2 U microscope and the Nikon NIS-elements software (Tokyo, Japan).
4
Quantifying Saos-2 Cell Proliferation
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Saos-2 cells were seeded in a Petri dish with a diameter of 35 mm (with a coverslip inside) at 1.5 × 105/mL, cultured for one day, and synchronized with medium containing 0.4% FBS for three days, leaving most cells in the G0 phase. Before the culture ended, BrdU (Thermo Fisher Scientific, USA; final concentration: 30 mg/L) was added for incubation at 37°C for 40 min. The medium was then discarded, and the coverslip was washed three times with PBS and fixed with methanol/acetic acid for 10 min. Endogenous oxidase was inactivated by 0.3% hydrogen peroxide-methanol for 30 min after air drying of the fixed coverslip. Subsequently, the coverslip was blocked with 5% normal rabbit serum (Abcam, USA), and nucleic acid was denatured with formamide at 100°C for 5 min. After being cooled on an ice-bath, the coverslip was washed with PBS, and anti-mouse BrdU monoclonal antibody (Abcam, USA; working concentration: 1:50) was added. Meanwhile, PBS or serum was added to negative control. The avidin–biotin complex (ABC) method was employed for detection. Hematoxylin and eosin (HE) staining was carried out. Total cells and BrdU-positive cells in 10 randomly selected high-magnification visual fields were counted under a light microscope (Olympus, Japan), and the labeling index was calculated.
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