Immunohistochemical staining was performed as described previously [14 (link)]. Briefly, IHC was performed using a PV-9001 kit (ZSGB-BIO) following the manufacturer’s instructions. The staining intensity was defined with a four-grade scoring system: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The staining extent was quantified as five value grades: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The sum of intensity and extent values was defined as the IHC score. The primary antibodies included anti-KDM6A antibody, anti-ARHGDIB antibody, and anti-Ki67 antibody (Invitrogen, PA5–19462).
Pv 9001 kit
Manufactured by ZSGB-BIO
The PV-9001 kit is a laboratory equipment designed for general research and analysis purposes. It includes a set of essential components to facilitate various experimental procedures. The product specifications and technical details are available upon request.
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4 protocols using pv 9001 kit
1
Immunohistochemical Staining Evaluation
2
Immunohistochemical Analysis of Target Protein
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H&E staining was performed according to standard methods. The expression of the target protein was assessed by IHC using a PV‐9001 Kit (ZSGB‐BIO). The IHC score was calculated as reported previously.31
3
Histological and Immunological Tissue Analysis
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Tissues were fixed in 4% paraformaldehyde (PFA), dehydrated in ethanol, embedded in paraffin wax, and sectioned (5 μm). To observe the morphology of tissues, sections were stained with the hematoxylin-eosin (HE). For immunohistochemistry, preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 28, 2021. ; https://doi.org/10.1101/2021.10.28.466222 doi: bioRxiv preprint sections were treated with 3% H2O2 for 20 minutes after antigen retrieval, followed by 1 hour in 5% bovine serum albumin (BSA). Sections incubated in primary antibodies overnight at 4 ℃ were subsequently treated with PV-9001 kits (ZSBIO) before being exposed to diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. For immunofluorescence, after antigen retrieval, sections were blocked in 5% bovine serum albumin or 5% donkey serum and then incubated in primary antibodies overnight at 4 ℃. Then the sections were washed in wash buffer and incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies at 37 ℃ for 1 h. After staining with DAPI (Vector laboratories), samples were analyzed using confocal microscopy (Zeiss LSM 780). Antibodies were in Supplement Table S3.
The copyright holder for this this version posted October 28, 2021. ; https://doi.org/10.1101/2021.10.28.466222 doi: bioRxiv preprint sections were treated with 3% H2O2 for 20 minutes after antigen retrieval, followed by 1 hour in 5% bovine serum albumin (BSA). Sections incubated in primary antibodies overnight at 4 ℃ were subsequently treated with PV-9001 kits (ZSBIO) before being exposed to diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. For immunofluorescence, after antigen retrieval, sections were blocked in 5% bovine serum albumin or 5% donkey serum and then incubated in primary antibodies overnight at 4 ℃. Then the sections were washed in wash buffer and incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies at 37 ℃ for 1 h. After staining with DAPI (Vector laboratories), samples were analyzed using confocal microscopy (Zeiss LSM 780). Antibodies were in Supplement Table S3.
4
Histological and Immunological Tissue Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were fixed in 4% paraformaldehyde (PFA), dehydrated in ethanol, embedded in paraffin wax, and sectioned (5 μm). To observe the morphology of tissues, sections were stained with the hematoxylin-eosin (HE). For immunohistochemistry, preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted October 28, 2021. ; https://doi.org/10.1101/2021.10.28.466222 doi: bioRxiv preprint sections were treated with 3% H2O2 for 20 minutes after antigen retrieval, followed by 1 hour in 5% bovine serum albumin (BSA). Sections incubated in primary antibodies overnight at 4 ℃ were subsequently treated with PV-9001 kits (ZSBIO) before being exposed to diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. For immunofluorescence, after antigen retrieval, sections were blocked in 5% bovine serum albumin or 5% donkey serum and then incubated in primary antibodies overnight at 4 ℃. Then the sections were washed in wash buffer and incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies at 37 ℃ for 1 h. After staining with DAPI (Vector laboratories), samples were analyzed using confocal microscopy (Zeiss LSM 780). Antibodies were in Supplement Table S3.
The copyright holder for this this version posted October 28, 2021. ; https://doi.org/10.1101/2021.10.28.466222 doi: bioRxiv preprint sections were treated with 3% H2O2 for 20 minutes after antigen retrieval, followed by 1 hour in 5% bovine serum albumin (BSA). Sections incubated in primary antibodies overnight at 4 ℃ were subsequently treated with PV-9001 kits (ZSBIO) before being exposed to diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin. For immunofluorescence, after antigen retrieval, sections were blocked in 5% bovine serum albumin or 5% donkey serum and then incubated in primary antibodies overnight at 4 ℃. Then the sections were washed in wash buffer and incubated with the appropriate Alexa-Fluor-conjugated secondary antibodies at 37 ℃ for 1 h. After staining with DAPI (Vector laboratories), samples were analyzed using confocal microscopy (Zeiss LSM 780). Antibodies were in Supplement Table S3.
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