Slides were obtained from Prince of Wales Hospital. Xylene and ethanol were used to remove the wax. Antigen retrieval was performed with sodium citrate buffer (Thermo Fisher Scientific) at 98°C for 15 min. Blocking was performed in 10% normal serum (goat and donkey, Abcam) with 1% BSA in PBST buffer (0.05% Triton X-100). IDH1(R132H) antibody (1:40; Dianova) and ADCY8 antibody (1:200; Abcam) were added to slides and incubated at 4°C overnight in a humid box. Secondary antibodies (1:400; anti-mouse, anti-rabbit; Thermo Fisher Scientific) were used to provide the fluorescent signal. The mounting buffer with DAPI (Abcam) was used to stain the nucleus and retain fluorescence. Images were taken with Zeiss Axio Scan.Z1 Slide Scanner with a ×20 objective (Zeiss).
Idh1 r132h antibody
Manufactured by Dianova
Sourced in Germany
The IDH1 R132H antibody is a laboratory reagent used for the detection and identification of the IDH1 R132H mutant protein. It is a specific and sensitive tool for research purposes, providing a reliable method to analyze the presence and distribution of this particular genetic alteration.
Automatically generated - may contain errors
Lab products found in correlation
Axio scan z1 slide scanner, by Zeiss (1 mentions)
Sodium citrate buffer, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Secondary hrp linked antibodies, by Santa Cruz Biotechnology (1 mentions)
Vinculin, by Abcam (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Ventana benchmark xt, by Roche (1 mentions)
5 protocols using idh1 r132h antibody
1
Immunofluorescence Staining of IDH1 and ADCY8
2
Western Blot Protein Expression Analysis
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Specific protein expression in cell lines was determined by western blot analysis as described before33 (link), 34 using the following primary antibodies: Mcl-1 (1:500; #5453 CST: Cell Signaling Technology, Danvers, MA), Bcl-2 (1:500; #4223 CST), mutation specific IDH1 R132H antibody (1:250; #DIA-H09 Dianova GmbH, Hamburg, Germany), human caspase-9 (1:1,000; #9502 CST), cleaved caspase-3 (1:250; #9664 CST), total PARP (1:1000; #9532 CST), cleaved PARP (Asp214, 1:1000; #9541 CST), Bcl-xL (1:500; #2764 CST), Bim (1:500; #2933 CST), Bak (1:500; #6947 CST) Noxa (1:500, clone 114C307; Calbiochem), β-actin (1:8,000, clone AC15; A1978 Sigma Aldrich), Vinculin (1:1000, #ab129002 Abcam). Secondary HRP-linked antibodies were purchased from Santa Cruz Biotechnology.
Capillary electrophoresis was performed on the Wes simple instrument (Protein simple, San Jose, CA) using the 12–230 kDa Wes separation module and anti-rabbit or anti-mouse detection modules according to the manufacturer’s instructions. Antibody concentrations are available on the Protein simple website. Uncropped western blot and capillary electrophoresis data are shown in Supplementary Fig.9 .
Capillary electrophoresis was performed on the Wes simple instrument (Protein simple, San Jose, CA) using the 12–230 kDa Wes separation module and anti-rabbit or anti-mouse detection modules according to the manufacturer’s instructions. Antibody concentrations are available on the Protein simple website. Uncropped western blot and capillary electrophoresis data are shown in Supplementary Fig.
3
Immunohistochemical Detection of IDH1 R132H
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Immunohistochemistry using a mouse monoclonal anti-human isocitrate dehydrogenase 1 (IDH1) R132H antibody (Dianova, Hamburg, Germany) (clone: H09, dilution 1:50) was performed using the EnVision FLEX system (Dako Japan Inc., Tokyo, Japan) (polymeric method), an automatic staining machine [21 (link)].
4
Screening for IDH1 Mutations
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Mutational screening for IDH1 was assessed with a mutation specific IDH1 antibody and by quantitative pyrosequencing. Staining for IDH1 R132H was carried out on 4 μm tissue sections from formalin-fixed, paraffin-embedded tumor specimens using the IDH1 R132H antibody (dianova GmbH, Germany). This antibody detects the heterozygous R132H point mutation of the IDH1 gene, resulting in a substitution of arginine to histidine at position 132 of the amino acid sequence. For the immunohistochemical reactions, paraffin sections were dried at 80°C for 15 min and stained with the primary IDH1 antibody with a dilution of 1:10 on a Ventana BenchMark XT® immunostainer (Ventana Medical Systems, Tucson, AZ) following the Ventana staining procedure. Immunoreaction was scored positive when tumor cells showed a strong cytoplasmatic and nuclear staining for IDH1 R132H. The presence of mutations affecting IDH1 codon 132 and IDH2 codon 172 were additionally assessed by pyrosequencing using the same reaction solutions, system and software as described above. The corresponding sequencing primers are given on request. Each tumor sample was analyzed in triplicates and each control sample in duplicates by individual PCR reactions. Non-neoplastic surgical biopsies of the white matter, obtained from 5 patients served as control.
5
Histopathological Diagnosis and Molecular Profiling of Brain Tumors
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After tumor removal or biopsy, histopathological diagnoses of all tumors were based on the WHO criteria published in 2016. [6] (link) As in our previously published method, [27] (link) expression of mutated isocitrate dehydrogenase 1 (IDH1) gene was evaluated by immunohistochemistry using the IDH1 R132H antibody (1:100 dilution; Dianova, Barcelona, Spain). If the result was negative, direct DNA sequencing was performed. The standard method of fluorescence in situ hybridization (FISH) was used to determine the 1p/19q deletion as published previously. [26] (link) The two-color FISH assay was performed on
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