Sections were routinely dewaxed and washed, after which they were stained by SP method. Antigens were retrieved with sodium citrate buffer. The remaining steps were performed according to the instructions of SP kit (ZSGB-BIO, China). Sections were incubated with mouse anti-GFAP polyclonal antibody (Cat. No. BM0055, 1:200, Bobst Biotechnology, China) overnight at 4 °C, following which Diaminobenzidine was used as a developer, and hematoxylin was used as a counterstain. Finally, they were observed under an optical microscope. Experimental procedures are explained in detail see in Additional file 7 . Each specimen was randomly selected in 5–6 different fields of view according to different hippocampal regions (CA1, CA3, DG) under a 40 × microscope to count the number of GFAP + cells. Under a 40 × microscope, 5 morphologically intact astrocytes were randomly selected from each specimen for Sholl analysis. The specific steps are as follows: GFAP signal was segmented with the threshold tool and converted to binary mask before its skeletonization with the skeletonize tool. The latter tool allowed to obtain segment length and any possible bifurcation of the skeletonized image analyzed with the Fiji-Image J software. Then, the number of synaptic junctions, synaptic end-point voxels, synaptic branches and the average branch length were measured with the Sholl plugin.
Sp kit
Manufactured by ZSGB-BIO
Sourced in China
The SP kit is a laboratory equipment designed for sample preparation. It provides a standardized and efficient method for processing biological samples prior to analysis. The core function of the SP kit is to facilitate the extraction, purification, and concentration of target analytes from complex matrices. The kit includes all necessary consumables and reagents to perform this sample preparation workflow.
Automatically generated - may contain errors
Lab products found in correlation
Zli 9018, by ZSGB-BIO (1 mentions)
Immunofluorescence microscope, by Olympus (1 mentions)
Spot image acquisition system, by Nikon (1 mentions)
Dab kit, by ZSGB-BIO (1 mentions)
K3513, by Merck Group (1 mentions)
Leitz dmrbe, by Leica (1 mentions)
Nanozoomer sq digital slide scanner, by Hamamatsu Photonics (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Dab staining kit, by ZSGB-BIO (1 mentions)
Ab15580, by Abcam (1 mentions)
Sc 7963, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Hif 1α, by GeneTex (1 mentions)
Pa110, by Tiangen Biotech (1 mentions)
Pv 9002, by ZSGB-BIO (1 mentions)
Cdk6 antibody, by Santa Cruz Biotechnology (1 mentions)
Dab chromogenic kit, by Beyotime (1 mentions)
20 protocols using sp kit
1
GFAP-Positive Astrocyte Characterization
2
Histological and Immunohistochemical Analysis
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Tumors and livers tissues were fixed in 4% PFA, embedded in paraffin, sectioned, and then stained. H&E-stained sections were independently evaluated at 40× and 100× magnification by two pathologists who were blinded to the experimental conditions. IHC-stained sections were processed with an SP Kit (Ovitalin-Biotin Detection System for Streptomyces Rabbits) purchased from ZSGB-BIO according to the manufacturer’s protocol. Positive signals were detected by using DAB color developing agent. Images were captured using a Leica microscope. The protein expression level was determined by measuring the mean densitometry and assessed with ImageJ software. By measuring the integrated optical density (IOD) and area of each image, the average optical density (mean density = IOD/area) was calculated, which reflects the per unit area concentration of the target protein. The signal density of the tissue areas was measured in at least three sections.
3
Keratin 7 Expression Analysis
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Strict serial 4 μm sections were cut from formalin-fixed paraffin-embedded tissue blocks. The detection of Keratin 7-positive cells was performed via staining the sections with a primary antibody against Keratin 7 using SP kit (SP-9002, ZSGB-BIO) according to the manufacturer’s instructions. Stains were detected using anti-immunoglobulin-coupled horseradish peroxidase with 3,3′-diaminobenzidine (DAB, ZLI-9018, ZSGB-BIO) as substrate. Nuclear counterstaining was performed with Cole hematoxylin (G1140, Solarbio).
4
Immunohistochemical Analysis of Endometrial Tissue
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The expressions of LIF, CB1, and FAAH in endometrial tissue were measured by immunohistochemical staining. The immunohistochemical staining was performed using anti-CB1 (Boster, Wuhan, China), anti-LIF (Bioss, Beijing, China) and anti-FAAH antibodies (Bioworld, USA), and SP kit (ZSGB-Bio, Beijing, China) according to the manufacturer’s instruction. Paraffin sections (4 mm) were cut from the endometrial tissue, deparaffinized in xylene, rehydrated in graded alcohol, and treated with hydrogen peroxide for 10 min to block endogenous peroxidases. After antigen retrieval by target retrieval solution for 10 min, tissue sections were blocked with goat serum for 20 min. The sections were then incubated with 50 µL anti-CB1, anti-LIF, and anti-FAAH antibodies at 4°C overnight. Slides were then incubated with HRP-conjugated streptavidin (SA-HRP) at 37°C for 20 min, developed in 3,3′-diaminobenzidine chromogen (DAB, ZSGB-Bio, Beijing, China) for 6 min, counterstained with Meyer’s hematoxylin and mounted. Exclusion of the primary antibody during immunostaining was used as a negative control. The integral optical density (IOD) of the specimens were determined by CMOS-based image sensor (Olympus, Tokyo, Japan) and analyzed for the relative quantity of positive reactant.
5
Immunohistochemistry of H. pylori Gastritis
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For immunohistochemistry, sections from C57BL/6 mice and 21 H. pylori- and 19 H. pylori+ gastritis patient gastric mucosa biopsies were deparaffinized and rehydrated, followed by antigen retrieval using citrate buffer (pH 6.1) for 20 min and were then stained with primary antibodies overnight at 4°C. Slides were washed and treated with the universal type SP kit and DAB kit (ZSGB-BIO) according to the manufacturer's instructions. Images were acquired using an immunofluorescence microscope (Olympus) equipped with a 20×objective, analyzed and processed by a Nikon & spot image acquisition system.
6
Immunohistochemical Analysis of KIF3A
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The expression level of KIF3A was detected by immunohistochemical staining, performed according to the instructions of the SP kit ZSGB-BIO, Beijing, China). The antibody for KIF3A was rabbit polyclonal anti-KIF3A (1:800; cat. no. K3513; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Results of the staining were evaluated separately by three pathologists under double-blind conditions and scored by the intensity of positive, the positive rates and final score (score of positive rates multiplied by score of intensity of positive). Detailed scoring system and criteria are presented in Table I .
7
Immunohistochemical analysis of 14-3-3ε and filamin A
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The immunohistochemical procedure was performed according to the general‐purpose SP kit (ZSGB‐BIO, China) instructions. Sections were incubated with anti‐14‐3‐3ε or antifilamin A at 4°C overnight, and each section was incubated with secondary antibody at room temperature for 30 minutes. For negative controls, the primary antibody was substituted with PBS. Each experiment was repeated three times, and images were taken using a microscope (Haimen Changlong, China). Semiquantitative integration method was adopted with modifications from Magara et al14 to determine the immunohistochemical staining positivity. Briefly, a value of 0, 1, 2, 3, or 4 was assigned according to the proportion of positive cells to the total cells in the observed field: less than 5%; 5%‐25%; 26%‐50%; 51%‐75%; and more than 75%, respectively. These values were then multiplied by the staining intensity of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow brown), or 3 (strong staining, brown) to obtain a score ranging from 0 to 12. A score equal to or <3 was considered low expression, and a score >3 was considered high expression. Each slide underwent 5 random counts using the high‐power field (400×), and the average was taken. The examiner was not blinded for the study, and informed consent was obtained from all localization patients.
8
Immunohistochemical Analysis of Biomarkers
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The antibodies used in the present study were as follows: Mouse monoclonal antibodies for E-cadherin and p53 (ZSGB-Bio, China), rabbit polyclonal antibodies for TGF-β1 and HIF-1α (Boster Biological Technology, China), mouse monoclonal antibody for N-cadherin (Santa Cruz, USA), and rabbit polyclonal antibodies for RB1CC1 (Proteintech Group, China). All antibodies used in the present study were suitable for the detection of proteins in both humans and mouse.
Immunohistochemistry was performed on paraffin sections. Deparaffinized sections were pretreated with 0.4% pepsin for 60 min at 37°C. Endogenous peroxidase activity was quenched by treatment with 0.2% H2O2 for 3 h. The sections were then incubated with the specific antibodies overnight at 4°C. In addition, sections incubated with 0.01 mol/l phosphate buffer saline (PBS) and tongue cancer tissues incubated with the chosen antibodies were used as negative and positive controls, respectively. The immunostaining was visualized with an SP kit (ZSGB-Bio, China) using a diaminobenzidine-peroxidase substrate. The sections were counterstained with Mayer's hematoxylin and examined using the image analyzer of a light microscope (Leica Leitz DMRB/E, Leica Microsystems, Wetzlar, Germany).
Immunohistochemistry was performed on paraffin sections. Deparaffinized sections were pretreated with 0.4% pepsin for 60 min at 37°C. Endogenous peroxidase activity was quenched by treatment with 0.2% H2O2 for 3 h. The sections were then incubated with the specific antibodies overnight at 4°C. In addition, sections incubated with 0.01 mol/l phosphate buffer saline (PBS) and tongue cancer tissues incubated with the chosen antibodies were used as negative and positive controls, respectively. The immunostaining was visualized with an SP kit (ZSGB-Bio, China) using a diaminobenzidine-peroxidase substrate. The sections were counterstained with Mayer's hematoxylin and examined using the image analyzer of a light microscope (Leica Leitz DMRB/E, Leica Microsystems, Wetzlar, Germany).
9
Immunohistochemical Protocol for p16 and PCNA
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After deparaffinization, hydration and antigen retrieval, sections were blocked for endogenous peroxidase and non-specific protein binding sites in a wet box containing a small volume of water using an SP kit (a detection system using rabbit streptavidin–biotin) (ZSGB, Beijing, China). Next, we gently removed the liquid from the slices. Primary antibodies were added to the sections and incubated at 4 °C overnight. The primary antibodies were rabbit anti-p16INK4a polyclonal antibody (1:200; Abcam, Cambridge, UK) and rabbit anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (1:500; Abcam, Cambridge, UK).
On the following day, the tissues sections were washed with phosphate-buffered saline (PBS, pH 7.4) three times for 10 min each. Then, biotin-labelled goat anti-rabbit IgG polymer was added to the sections, which were incubated for 15 min and washed with PBS. This was followed by addition of HRP-labelled streptavidin for 15 min. The sections were washed with PBS, stained at room temperature with diaminobenzidine for 3–5 min, counterstained, and covered. Sections were observed and images were collected with a Nano Zoomer-SQ digital slide scanner (Hamamatsu, Japan).
On the following day, the tissues sections were washed with phosphate-buffered saline (PBS, pH 7.4) three times for 10 min each. Then, biotin-labelled goat anti-rabbit IgG polymer was added to the sections, which were incubated for 15 min and washed with PBS. This was followed by addition of HRP-labelled streptavidin for 15 min. The sections were washed with PBS, stained at room temperature with diaminobenzidine for 3–5 min, counterstained, and covered. Sections were observed and images were collected with a Nano Zoomer-SQ digital slide scanner (Hamamatsu, Japan).
10
Immunohistochemical Analysis of Tumor Sections
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Tumors were fixed by 4% paraformaldehyde, paraffin embed and sliced into sections, sections were hydrated by xylene and gradient alcohol (Sinoreagent, China). Antigen of sections were repaired by citrate solution and blocked by goat serum, then sections incubated with primary and secondary antibodies according to the manufacturer’s protocol of SP Kit (ZSGB-BIO, China) and stained by DAB Staining Kit (ZSGB-BIO, China) and hematoxylin solutions (Sinoreagent, China). The pictures of sections were photographed by inverted microscope (Olympus, Japan). Relative staining score was calculated using an IHC score analysis method according to the proportion of positively stained cells and the intensity of staining. The proportion of positive cells was scored as follows: 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), 4 (> 75%) and the intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong).
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