IHC was performed on 5-μm-thick formalin-fixed paraffin-embedded slices. After deparaffinization, rehydration, antigen retrieval and endogenous peroxidase inhibition, the samples were incubated with anti-NSD2 (Abcam), anti-TUNEL (Roche) and anti-cleaved caspase 3 (Servicebio) antibodies at 4 °C for 8–10 h. After the secondary antibody was applied, diaminobenzidine (DAKO) solution was used as a chromogen. Furthermore, haematoxylin (Sigma) staining was performed to identify nuclei. Images were acquired using a microscope (Leica DM 4000B).
Anti nsd2
Manufactured by Abcam
Anti-NSD2 is a laboratory reagent used in research applications. It is a polyclonal antibody that specifically binds to and detects the NSD2 protein. NSD2 is a histone methyltransferase involved in the regulation of gene expression. This antibody can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to study the expression and localization of NSD2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dab kit, by Beijing Strong Biotechnologies (2 mentions)
Anti cleaved caspase 3, by Wuhan Servicebio Technology (1 mentions)
Anti tunel, by Roche (1 mentions)
Dm4000b, by Leica (1 mentions)
Haematoxylin, by Merck Group (1 mentions)
Anti h3k36me2, by Active Motif (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Anti ep300, by Abcam (1 mentions)
Anti h4k16ac, by Abcam (1 mentions)
Anti h4k5ac, by Abcam (1 mentions)
Anti h4k8ac, by Abcam (1 mentions)
Anti wt1, by Abcam (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
5 protocols using anti nsd2
1
Immunohistochemical Analysis of NSD2, TUNEL, and Caspase-3
2
Evaluation of NSD2 Protein Expression in ccRCC
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The expression of NSD2 protein in 65 ccRCC and 10 normal tissue sections were examined by IHC. Samples were obtained from patients with local or locoregionally advanced disease, who had never received any previous treatment. Slides of paraffin-embedded tissues were deparaffinized, rehydrated, and sections were treated at 96℃ for 20 minutes in antigen retrieval buffer. Anti-NSD2 (Abcam, dilution: 1:200) antibody was applied on tissue sections for 1 h incubation at room temperature. Then, the immune complexes were detected by using a DAB kit (MXB Biotechnologies, China). Semi-quantitative analysis of NSD2 staining using a 3-grade scale was defined as follows: negative, grade 0; mild, grade +1; and strong staining intensity, grade +2. Application of tissues was approved by the Ethics Committee of The Third Affiliated Hospital of Soochow University.
3
Chromatin Immunoprecipitation of H3K36me2 and NSD2
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Control and NSD2-KD1 143B cells (2 × 107 cells) were crosslinked, lysed and sheared using a UCD-300 (Bioruptor) to ~200–700 base pairs in length. ChIP was then performed using an EZ ChIP Kit (Millipore; 17-371) according to the manufacturer’s instructions. ChIP-enriched DNA was then quantified by RT-qPCR. The ChIP antibodies used were anti-H3K36me2 (Active Motif) and anti-NSD2 (Abcam). The primer sequences are listed in Table S3 .
4
Evaluating NSD2 Expression in ccRCC
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Between July 2018 and December 2018, a total of 30 specimens from 30 patients who had never accepted chemotherapy, radiotherapy or biotherapy were collected in the Department of Urology, the Third Affiliated Hospital of Soochow University. The patients were informed of the study and asked to sign informed consent. Application of tissues was approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University. The specimens were pathologically confirmed as ccRCC according to the WHO for the classification criteria.
The NSD2 protein level in ccRCC and adjacent normal tissues were evaluated by IHC. The paraffin-embedded specimens were deparaffinized, rehydrated, and treated in antigen retrieval buffer at 96°C for 20 mins. Then, the samples were incubated with anti-NSD2 (Abcam, dilution: 1:200) primary antibody at room temperature for 1 hr. The DAB kit (MXB Biotechnologies, China) was used for detecting immune complexes. One-renal cancer pathologist performed semi-quantitative analysis of NSD2 staining using a 4-grade scale was defined as follows: Sections with no labeling or labeled cells <5% were scored as 0. Sections with 5–30% of labeled cells were scored as +1, with 31–70% of labeled cells as +2, and with >70% of labeled cells as +3.
The NSD2 protein level in ccRCC and adjacent normal tissues were evaluated by IHC. The paraffin-embedded specimens were deparaffinized, rehydrated, and treated in antigen retrieval buffer at 96°C for 20 mins. Then, the samples were incubated with anti-NSD2 (Abcam, dilution: 1:200) primary antibody at room temperature for 1 hr. The DAB kit (MXB Biotechnologies, China) was used for detecting immune complexes. One-renal cancer pathologist performed semi-quantitative analysis of NSD2 staining using a 4-grade scale was defined as follows: Sections with no labeling or labeled cells <5% were scored as 0. Sections with 5–30% of labeled cells were scored as +1, with 31–70% of labeled cells as +2, and with >70% of labeled cells as +3.
5
Antibody Panel for Multimodal Protein Analysis
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Antibodies used for western blot (WB), immunofluorescence (IF) and chromatin immunoprecipitation (ChIP) were as follows: anti-H3K36me3 (Solarbio no. K003337P, 1:400 for IF), anti-H3K36me2 (Solarbio no. K003336P, 1:400 for IF), anti-H3 (Solarbio no. K107283P, 1:800 for WB), anti-γ-H2A.X (Solarbio no. K006207P, 1:200 for IF, 1:1000 for WB), anti-mCherry (Solarbio no. K200015M, 1:1000 for WB), anti-H4K16ac (Abcam no. ab109463, 1:200 for IF), anti-NSD2 (Abcam no. ab-75359, 1:1000 for WB, 1:200 for IF), anti-PSMA8 (Proteintech no. 4022-1-AP, 1:1000 for WB), anti-GAPDH (Proteintech no. 60004-1-Ig, 1:1000 for WB), anti-PRM2 (Briar Patch Biosciences, Hup2B, 1:200 for IF, 1:800 for WB), anti-WT1 (Abcam no. ab89901, 1:200 for IF), anti-H4K5ac (ABclonal no. A19525, 1:1000 for WB, 1:200 for IF), anti-H4K8ac (ABclonal no. A7258, 1:1000 for WB, 1:200 for IF), anti-EP300 (Abcam no. ab-10485, 1:1000 for WB), anti-H4 (ABclonal no. A1131, 1:1000 for WB) and anti-α-Tubulin (Proteintech no. 66031-1-Ig, 1:1000 for WB).
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