Total proteins were extracted from transfected or non-transfected cells, loaded (50 μg per lane) on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were blocked for 2 h at 37°C with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) and then incubated overnight at 4°C with the following primary antibodies: CDK1 (ab133327, Abcam, Cambridge, UK), cyclin B1 (ab18250; Abcam), cyclin A2 (18202-1-AP; Proteintech, Wuhan, China), MLH1 (ab92312; Abcam), PMS2 (ab110638; Abcam), MSH2 (ab92473; Abcam), MSH6 (ab92471; Abcam) and GAPDH (T0004; Affinity, Changzhou, China). Then, the membranes were incubated with an HRP-conjugated secondary antibody for 1 h at 37°C and covered with ECL luminescence reagent (Perkin-Elmer Inc., Waltham, MA, USA). GAPDH was used as the internal control.
Ab92471
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab92471 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory techniques, such as immunohistochemistry, immunocytochemistry, and Western blotting. The antibody targets a specific antigen, but further details on its intended use or application are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab92312, by Abcam (12 mentions)
Ab110638, by Abcam (9 mentions)
Ab70270, by Abcam (3 mentions)
Ab227941, by Abcam (3 mentions)
Ab15580, by Abcam (2 mentions)
Ab200828, by Abcam (2 mentions)
Ab109185, by Abcam (2 mentions)
Ab212188, by Abcam (2 mentions)
Ab207718, by Abcam (2 mentions)
Ab3982, by Abcam (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Envision dual link hrp, by Agilent Technologies (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
T0004, by Affinity Biosciences (1 mentions)
Ab18250, by Abcam (1 mentions)
Ab92473, by Abcam (1 mentions)
Ab133327, by Abcam (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Ab109413, by Abcam (1 mentions)
Goat anti rabbit mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
14 protocols using ab92471
1
Western Blot Analysis of Cell Cycle and DNA Repair Proteins
2
Immunohistochemistry Analysis of LY6G6D and MMR Proteins
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Immunohistochemistry (IHC) analysis was performed as previously reported [8 (link)] using anti-LY6G6D (ab139649 Abcam, Cambridge, UK) on 4-μm-thick histological TMA sections. Mismatch repair (MMR) was investigated using the following antibodies: human mutS homolog 2 (anti-MSH2, ab92372), MutL homolog 1 (anti-MLH1, ab92312), human mutS homolog 6 (anti-MSH6, ab92471) from (Abcam Cambridge, UK). LY6G6D positive intrastromal cells were counted automatically with ImageJ-based software. All the cell counts were expressed as cells mm−2 as already reported [8 (link)]. The proportion of cancer cells stained was scored regardless of intensity as follows: Negative, as the complete absence of staining in more than 95% of tumor cells; Weak/moderate, characterized by a limited number of tumor cells scattered in a background of either negative or positive tumor cells; High or intense as a homogeneous staining in virtually all tumor cells.
3
Immunohistochemical Analysis of DNA Repair Proteins
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Immunostains for Chk2 protein expression were performed on 4 μm sections from colon tumor and normal mucosa from family K and CRC control or healthy control. After deparaffinization, citrate buffer was used to retrieve antigen. Endogenous peroxidase was inactivated with 3% H2O2 dilution for 15 minutes. Tissues were blocked with 10% goat serum for 1 hour at room temperature. The Chk2 primary antibody (ab109413, diluted at 1:100, Abcam) was incubated overnight at 4°C. The goat anti-rabbit/mouse secondary antibody (Thermo Fisher Scientific, D-3004) was incubated for 1 hour at room temperature and subsequently revealed with DAB substrate (Dako) for 3 minutes. Slides were finally stained in hematoxylin. Immunostains for MSH2 (Thermo Fisher Scientific, diluted at 1:150, 33-7900), MSH6 (Abcam, diluted at 1:150, ab92471), PMS2 (Abcam, diluted at 1:100, ab110638), and MLH1 (Abcam, diluted at 1:100, ab92312) proteins expressions were performed as described above.
4
Antibody Profiling for Cancer Markers
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The following antibodies were purchased from the respective suppliers: anti-RNPS1 (ab79233), anti-Ki-67 (ab15580), anti-CEA (ab207718), anti-CA199 (ab3982), anti-CA153 (ab109185), anti-HE4 (ab200828), anti-Bcl-2 (ab182858), anti-Bax (ab182733), anti-MLH1 (ab92312), anti-cleaved caspase-3 (ab2302), anti-MSH2 (ab212188), anti-MSH6 (ab92471), and anti-PMS2 (ab110638) (all from Abcam, Cambridge, USA) and anti-β-actin (M01263-2; Boster, Wuhan, China). The secondary antibodies used were anti-rabbit IgG (AS014) and anti-mouse IgG (H+L) (AS003), both from ABclonal (Wuhan, China), and IMR-1A (HY-100431A; MedChemExpress, Dallas, TX, USA).
5
Protein Extraction and Western Blot Analysis
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Proteins were extracted by solubilizing the cells in boiling SDS buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 1% SDS). Samples were boiled for 5 min at 95 °C and sonicated for 10 s. Extracts were clarified by centrifugation, normalized with the BCA Protein Assay Reagent kit (Thermo). Equal amounts of proteins (20 μg) were loaded in each lane. Proteins were separated by PAGE and transferred to nitrocellulose sheets. Western blot detection was performed with enhanced chemiluminescence system (GE Healthcare) and peroxidase-conjugated secondary antibodies (Amersham). The following primary antibodies were used for western blotting: anti-beta2 Microglobulin [EP2978Y] (ab75853, Abcam), anti-MLH1 (ab92312, Abcam), anti-MSH2 (ab70270, Abcam), anti-MSH6 [EPR3945] (ab92471, Abcam), anti-MSH3 PA527864, Invitrogen, anti-PMS2 EPR3947 (Cell Marque Corporation, USA), anti-actin (I-19) (sc1616, Santa Cruz), and anti-HSP 90α/β (H-114, sc-7947, Santa Cruz). Images were acquired with Chemidoc (Biorad), and western blot band intensity was analyzed using Image Lab software (Biorad).
6
Immunohistochemical Analysis of Colorectal Tumors
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At necropsy, blood was collected and the SI, LI, caecum, stomach, liver, lungs, spleen, thymus, lymph nodes, and any other organ or tissue showing abnormalities were collected and fixed in 10% neutral‐buffered formalin for 24 h. Tissues were processed and paraffin‐embedded to blocks, sectioned, and stained with haematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed using anti‐MSH2 (ab70270, 1:4000; Abcam, Cambridge, UK), anti‐MSH6 (ab92471, 1:500, Abcam), anti‐MLH1 (ab92312, 1:250, Abcam), anti‐Ki‐67 (ab16667, 1:500, Abcam), anti‐β‐catenin (610154, 1:100; BD Transduction Laboratories, San José, CA, USA), anti‐cCas3 (9664, 1:50; Cell Signaling Technologies, Danvers, MA, USA), anti‐γH2AX (2577, 1:100, Cell Signaling Technologies), and anti‐p53 (CM5, 1:100; provided by Dr Phil Coates, Masaryk Memorial Cancer Institute, Brno, Czechia) antibodies, as previously described [22 (link)]. Image analysis software QuPath v0.2.0 was used to analyse immunohistochemically detected MSH2, Ki67, γ‐H2AX, and p53 protein expression [25 (link)].
7
MMR Proteins Immunohistochemistry
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Antibodies against MMR proteins performed in this study were the following: rabbit monoclonal anti-MLH1 antibody (dilution 1:100, #ab92312) (Abcam, Cambridge, MA, USA), mouse monoclonal anti-MSH2 antibody (dilution 1:50, #33-7900) (Invitrogen, Carlsbad, CA, USA), rabbit monoclonal anti-MHS6 antibody (dilution 1:100, #ab92471) (Abcam, Cambridge, MA, USA) and anti-PMS2 antibody (dilution 1:25, #ab110638) (Abcam, Cambridge, MA, USA).
8
Endometrial Cell Line Characterization
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This study used normal endometrial cells (NECs), KLE cells, RL952 cells, Ishikawa cells, and ECC-1 cells. They were maintained and cultured as previously described [15 (link)]. The cell lines used in this study were as follows: normal endometrial cells (NEC, CP-H058, Procell, Wuhan, China), KLE (CL-0133, Procell, Wuhan, China), RL952 (CL-0197, Procell, Wuhan, China), Ishikawa (CL-0283, Procell, Wuhan, China), and ECC-1 (BS-C163325, BinSuiBio, Shanghai, China).
The following antibodies were used: Anti-METTL5 (16791-1-AP, Proteintech, Rosemont, USA), anti-Ki-67 (ab15580, Abcam, Cambridge, USA), anti-CEA (ab207718, Abcam, Cambridge, USA), anti-CA199 (ab3982, Abcam, Cambridge, USA), anti-CA153 (ab109185, Abcam, Cambridge, USA), anti-HE4 (13, ab200828, Abcam, Cambridge, USA), anti-MLH1 (ab92312, Abcam, Cambridge, USA), anti-MSH2 (ab212188, Abcam, Cambridge, USA), anti-MSH6 (ab92471, Abcam, Cambridge, USA), anti-PMS2 (ab110638, Abcam, Cambridge, USA), and anti-β-actin (M01263-2, Boster, Wuhan, China).
The following antibodies were used: Anti-METTL5 (16791-1-AP, Proteintech, Rosemont, USA), anti-Ki-67 (ab15580, Abcam, Cambridge, USA), anti-CEA (ab207718, Abcam, Cambridge, USA), anti-CA199 (ab3982, Abcam, Cambridge, USA), anti-CA153 (ab109185, Abcam, Cambridge, USA), anti-HE4 (13, ab200828, Abcam, Cambridge, USA), anti-MLH1 (ab92312, Abcam, Cambridge, USA), anti-MSH2 (ab212188, Abcam, Cambridge, USA), anti-MSH6 (ab92471, Abcam, Cambridge, USA), anti-PMS2 (ab110638, Abcam, Cambridge, USA), and anti-β-actin (M01263-2, Boster, Wuhan, China).
9
Immunohistochemical Evaluation of MMR Proteins
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All polyps were immunohistochemically stained with monoclonal antibodies against MLH1 (BD Pharmingen 551091, 1:40; BD Pharmingen, San Jose, CA, USA), PMS homologue 2 (PMS2) (BD Pharmingen 556415, 1:50), MutS homologue 2 (MSH2) (Calbiochem NA26, 1:40; Calbiochem, Darmstadt, Germany) and MSH6 (Abcam ab92471, 1:500; Abcam, Cambridge, UK) and developed with Powervision (LabVision, Cheshire, UK). We scored staining as positive, negative or unclear, as previously described.21 When mismatch repair (MMR) protein staining was negative or unclear, we tested MSI using a panel of microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26 and BAT40). MSI status was characterised as MSI when more than one marker was unstable and microsatellite stable (MSS) with one or no unstable markers.
10
Mismatch Repair Protein IHC in UTUC
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A total of 175 individual cases with formalin-fixed paraffin-embedded (FFPE) tumor blocks were collected. All hematoxylin and eosin-stained slides were reviewed by two professional pathologists. Tissue microarrays (TMAs) of 175 UTUC cases were constructed from the most typical regions of each case. MMR protein IHC was applied to detect the expression of the DNA MMR proteins MLH1 (#ab92312, Abcam, UK), PMS2 (#ab110638, Abcam, UK), MSH2 (#ab227941, Abcam, UK), and MSH6 (#ab92471, Abcam, UK). MMR proteins were considered lost when the tumor nuclei completely lacked staining and a positive internal control was present in the form of lymphocytes and/or endothelial cells.
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