Paraffin-embedded tissue blocks were used for immunohistochemical staining. Paraffin-embedded tissue specimens were sectioned, deparaffinized in xylene, and rehydrated, followed by antigen retrieval in sodium citrate, and the sections were then processed using SPlink Detection Kits (ZSGB-BIO) according to the manufacturer’s instructions. The sections were incubated with primary antibodies (1:200 dilution) overnight at 4°C. Specimens were stained using a DAB kit (ZSGB-BIO) until the desired stain intensity was developed. Sections were then counterstained with hematoxylin, dehydrated, and mounted. Staining intensity and extent of staining were graded as follows: negative (score 0), bordering (score 1), weak (score 2), moderate (score 3), and strong (score 4). Extent of staining was also grouped into quantiles according to the percentage of high-staining cells per field: negative (score 0), 25% (score 1), 26%–50% (score 2), 51%–75% (score 3), and 76%–100% (score 4). All immunohistochemical staining was evaluated and scored by at least 2 independent pathologists. Details on the Critical Commercial Assays are provided in Supplemental Table 3 .
Splink detection kit
Manufactured by ZSGB-BIO
Sourced in China
The SPlink Detection Kits are used for the detection and quantification of specific target molecules in biological samples. The kits utilize a specialized detection method to provide accurate and reliable results. This equipment is designed for use in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by ZSGB-BIO (5 mentions)
Bx600 microscope, by Olympus (4 mentions)
Spot flex camera, by Olympus (4 mentions)
Dab kit, by ZSGB-BIO (4 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
In situ cell death detection kit, by Roche (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Fluorescence microscope, by Zeiss (2 mentions)
Anti cd24, by Abcam (2 mentions)
Anti cd44, by Abcam (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Dab reagent kit, by ZSGB-BIO (2 mentions)
Optical microscope, by Olympus (2 mentions)
Anti hnrnp 1, by Santa Cruz Biotechnology (2 mentions)
Anti hnrnp l, by Santa Cruz Biotechnology (2 mentions)
Cellsens standard 1, by Olympus (2 mentions)
Anti cd45 antibody, by BioLegend (1 mentions)
Inverted phase contrast fluorescence microscope, by Olympus (1 mentions)
Phdr1, by Merck Group (1 mentions)
52 protocols using splink detection kit
1
Immunohistochemical Staining of Paraffin-Embedded Tissues
2
IHC Evaluation of LYN in Tissue Microarrays
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IHC evaluation of LYN was performed on the tissue microarray. IHC was preformed according to the Streptavidin/Peroxidase kit instructions (SPlink Detection Kits, ZSGB-BIO, China). After deparaffinized and rehydrated, the sections were subjected to heat in citrate buffer –induced antigen retrieval for 15 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 15 min. Thereafter, sections were blocked with goat serum for 15 min. Anti-LYN (1:100) was incubated overnight at 4°C. Then sections were incubated with HRP-conjugated secondary antibodies for 10 min and incubated in horseradish enzyme-labeled chain avidin solution for 10 min at 37°C and reacted with a DAB Horseradish Peroxidase Color Development Kit and counterstained with hematoxylin. Staining intensity was graded on a 0–3 scale as follows: 0 (absence of staining), 1 (weakly stained), 2 (moderately stained), and 3 (strongly stained). The percentage of positive tumor cells was scored as follows: 0 (absence of tumor cells), 1 (<33% tumor cells), 2 (33–66% tumor cells) and 3 (>66% tumor cells). Immunohistochemical score (ranging from 0 to 9) was calculated by multiplying the intensity score and the percentage score [27 (link)]. The same qualified pathologist analyzed all the IHC data to ensure consistency.
3
Immunohistochemical Analysis of Claudin-3 and CD45 in Colon Tissues
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Colon tissues were fixed in 4% paraformaldehyde in PBS, and the fixed sections were incubated in 3% H2O2 solution in PBS at room temperature for 10 min. Antigen retrieval was performed in sodium citrate buffer (0.01 M, pH 6.0) in a microwave oven at 1000 W for 3 min. Nonspecific antibody binding was blocked by incubation with 5% normal goat serum in PBS for 1 h at 25 °C. Slides were stained overnight at 4 °C with anti-claudin-3 antibody (Invitrogen, 34–1700, 1:200 dilution) and anti-CD45 antibody (BioLegend, 103102, 1:1000 dilution). The slides were subsequently washed and incubated with biotin-conjugated secondary antibodies for 30 min, and then with horseradish peroxidase streptavidin (HRP Streptavidin) for 30 min (SPlink Detection Kits; ZSGB-BIO, SP-9001 or SP-9002). The sections were developed using a 3,3’-Diaminobenzidine (DAB) substrate kit (ZSGB-BIO, ZLI-9017) and counterstained with hematoxylin. Images were captured using an Olympus BX600 microscope and a SPOT Flex camera. ImagePro Plus was used for further quantification of the DAB intensity in the image.
4
Immunohistochemistry and F-Actin Analysis
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Samples were blocked with 10% goat serum and incubated with the Ab at 4°C overnight. SPlink Detection Kits (ZSGB‐BIO) were used for IHC according to the manufacturer’s instructions. The slides were observed and photographed with an Olympus microscope, and the images were analyzed by ImageJ to measure the percentage of the positive zone. Five random fields were selected for analysis.
For F‐actin rings observation, cells were fixed with 4% paraformaldehyde, blocked with 10% goat serum, and incubated with rhodamine‐phalloidin (1:200, PHDR1; Sigma‐Aldrich) for 15 minutes. Next, the slides were mounted with a mounting medium and stained with DAPI (ZSGB‐BIO), after which the at least five random fields were photographed with an inverted phase‐contrast (fluorescence) microscope (Olympus).
For F‐actin rings observation, cells were fixed with 4% paraformaldehyde, blocked with 10% goat serum, and incubated with rhodamine‐phalloidin (1:200, PHDR1; Sigma‐Aldrich) for 15 minutes. Next, the slides were mounted with a mounting medium and stained with DAPI (ZSGB‐BIO), after which the at least five random fields were photographed with an inverted phase‐contrast (fluorescence) microscope (Olympus).
5
Immunohistochemical Staining Protocol for CD31
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Immunohistochemistry was performed using SPlink Detection Kits (SP 9002, ZSGB-BIO). The sections were fixed in acetone (4°C) for 10 min, and the antigens were retrieved in citrate buffer (0.01 M, pH 6.0, 99°C) for 10 min. Endogenous peroxidase activity was inhibited by incubating the sections with 3% hydrogen peroxide for 15 min, and nonspecific binding sites were blocked by incubating the sections with normal goat serum for 30 min at 37°C. Then, anti-mouse CD31 primary antibody (Abcam, UK) was added to the sections at a dilution of 1:500 in PBS [68 (link)]. The sections were incubated overnight at 4°C and then rewarmed at 37°C for 1 hour. The goat anti-mouse IgG secondary antibody solution was added, and the sections were incubated at 37°C for 30 min. Then, streptavidin-horseradish peroxidase (S-HRP) was added and incubated with the sections for 30 min. Subsequently, the sections were transferred to a diaminobenzidine (DAB) solution and immersed for approximately 1 min. Finally, the sections were dehydrated by sequential immersion in a gradient of ethanol solutions (70%, 80%, 90%, 100%, 100%, 100%; 10 min each) and cleared in xylene (3 × 10 min).
6
Immunohistochemical and Immunofluorescence Analysis
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IHC analysis was performed on 4 μm-thick sections using SP Link Detection Kits (ZSGB-BIO, Beijing, China), according to the manufacturer’s instructions. Briefly, sections were blocked with PBS containing 10% normal goat serum for 30 min, and subsequently incubated with primary antibodies, Col2 (diluted 1:1000), Mmp3 (diluted 1:100), Mmp13 (diluted 1:100), Nlrp3 (diluted 1:100), Caspase-1 (diluted 1:100), Gsdmd (diluted 1:100), IL-1β (diluted 1:100), IL-18 (diluted 1:100) at 4 ℃ overnight, respectively. The polymer-HRP labeled secondary antibody was added for 30 min the following day. The immunohistochemical reaction was visualized by incubation with 0.05% diaminobenzidine (DAB). Positive staining appeared in brown. Images were captured with a microscope (Carl Zeiss, Göttingen, Germany). For IF assay, sections were incubated with primary antibodies, including Cgrp (diluted 1:200), Netrin-1 (diluted 1:200), p-i-kbα (diluted 1:200), p65 (diluted 1:200) at 4℃ overnight, then treated with fluorescent-conjugated secondary-antibody (Sungene Biotech, Tianjin, China) for 30 min in the dark. After DAPI counterstaining, the images were captured with a fluorescence microscope (Carl Zeiss, Göttingen, Germany). The number of positive cells was quantified in a blinded manner with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) as we previously described.38 (link)
7
Histopathological Evaluation of Tumors
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Tumors were fixed in 10% neutral buffered formalin and, embedded with paraffin. 5 μm sections were cut and stained with hematoxylin and eosin (H&E). Immunohistochemical analyses for Ki67 were performed using SPlink Detection Kits (ZSGB-BIO, Beijing, China). Stained sections were examined using a Nikon Observer microscope.
8
Immunohistochemical Analysis of YAP1 Expression
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The formalin‐fixed and paraffin‐embedded tissues from postoperative patients were cut into 5‐μm sections. Sections were deparaffinized in xylene and rehydrated using a series of graded alcohols. Then slides were subjected to antigen retrieval with 0.01 m sodium citrate (pH 6.0) and blocking with 10% goat serum. The samples were incubated at 4 °C overnight with YAP1 (#12395; Cell Signaling) antibody. Immunostaining was performed using a SPlink Detection Kits (SP‐9002; ZSGB‐Bio, Beijing, China) according to the manufacturer's instructions. The percentage of positive cells was graded as per the following criteria: 0, < 10%; 1, 10–30%; 2, 31–50%; 3, more than 50%.
9
Histological and Immunohistochemical Analysis of Lung Tumors in Mice
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Lung lobes of tumor-bearing mice subjected to various treatments were fixed in 4% paraformaldehyde overnight and embedded in paraffin following standard procedures. Tissue sections (4 μM) were dewaxed and rehydrated. For histological analysis of tumor burden in mice, lung tissue sections were stained with hematoxylin and eosin (H&E) to define tumor tissue areas in the lung. For immunohistochemical analysis of specific antigens in the lung, the tissue sections were heated at 120 °C for 5 min using a pressure cooker in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval. The sections were then treated with 3% H2O2 solution in PBS at room temperature for 10 min to block endogenous peroxidase activity and further blocked with 5% normal goat serum for 1 h at room temperature. The sections were incubated with anti-MHCII antibodies (Invitrogen, catalog no. 14-5321-82) overnight at 4 °C. The tissue sections were subsequently washed with PBS and incubated with biotin-conjugated secondary antibodies for 30 min and then with horseradish peroxidase streptavidin (HRP streptavidin) for 30 min (SPlink Detection Kits; ZSGB-BIO, SP-9001). The sections were developed using the 3,3ʹ-diaminobenzidine (DAB) substrate kit (ZSGB-BIO, ZLI-9017) and counterstained with hematoxylin. Images were captured using an Olympus BX600 microscope and SPOT Flex camera.
10
Immunohistochemical Analysis of CD138 and HOXA10
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Briefly, the paraffin sections were dried at 60°C for 2 h and deparaffinized by xylene for 40 min. Then, the sections were dehydrated with gradient alcohols and subjected to antigen retrieval. Next, the sections were washed with PBS three times, treated with endogenous peroxidase blocker (SPlink Detection Kits, ZSGB-BIO) and incubated at 37°C for 10 min. Subsequently, the sections were washed with PBS three times and blocked with goat serum for 20 min at 37°C. The sections were incubated with primary antibody, including anti CD138 (1:200; Bioss) and anti HOXA10 (1:200; Bioss), at 4°C overnight. After being rewarmed at 37°C for 30 min and washed with PBS three times, the sections were incubated with the secondary antibody at 37°C for 20 min. Then 3,3′-diaminobenzidine staining was added to enable visualization. Three random HPFs were selected from each section under 200× magnification, and the images were analyzed by ImageJ software.
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