Ab92312

Immunofluorescence staining was performed as described in the previous studies (Yoon et al., 2018 (link)). The primary antibodies used in this study included as follows: mouse anti-SCP3 (1:600, ab97672, Abcam), rabbit anti-SCP1 (1:600, ab15090, Abcam), rabbit anti-γH2AX (1:300, ab26350, Abcam), and rabbit anti-MLH1 (1:200 ab92312, Abcam). The secondary antibodies used were goat anti-rabbit Alexa 488/594 (1:300, ab150077/ab150080, Abcam) and goat anti-mouse Alexa 488/594 (1:300, ab150113/ab150116, Abcam). Nuclear DNA was stained by 4′,6-diamino-2-phenylindole (DAPI). Images were captured by the fluorescence microscopy Imager M2 (ZEISS, Gottingen, Germany) at 100 × oil objective magnification and an Axiocam 503 monochrome camera.

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.

Expression of Mfn2 or Mlh1 in the mitochondria was performed in HRECs by immunofluorescence technique using antibodies against Mfn2 (Cat. No. ab56889, Abcam; 1:250 dilution) and Mlh1 (cat. no. ab92312, Abcam, Cambridge, MA, USA; 1:100 dilution), as previously described^{13 (link),14 (link)}. CoxIV was used as a mitochondrial marker; Cat. No. ab153709 for Mfn2 and Cat. No. ab33985 for Mlh1; both from Abcam, and at 1:250 dilution each. Secondary antibodies included Alexa Fluor-488 (green) conjugated anti-rabbit (Cat. No. Molecular Probes-Life Technologies, Grand Island, NE), DyLight 488-conjugated anti-mouse (Cat. No. DI-2488, Vector Laboratories, Burlingame, CA) and Texas red-conjugated anti-mouse (Cat. No. TI-2000, Vector Laboratories, Burlingame, CA); each at 1:500 dilution. Immuno-labelled cells were mounted using DAPI-containing (blue) Vectashield mounting medium (Vector Laboratories), and were examined under ZEISS (Carles Zeiss, Inc., Chicago, IL, USA) at 40X objective magnification with the Apotome module^{13 (link),14 (link)}. The images were calibrated with the ZEISS proinbuilt software package and modules, and the Pearson’s correlation coefficient was calculated using the colocalization software module^{13 (link)}.

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).

Paraffin-embedded tissue sections were deparaffinized and antigen retrieval was carried out using citrate buffer (pH = 6.0). Hydrogen peroxide was used for quenching the endogenous peroxide. IHC for O⁶-methylguanine-DNA methyltransferase (MGMT), MutS homolog 6 (MSH6), MutS homolog 2 (MSH2), MutL homolog 1 (MLH1), and postmeiotic segregation increased 2 (PMS2) was carried out on 21 tissue specimens of 29 operated patients. The tissue sections were incubated with primary antibodies for MGMT (MT 3.1, 1:100, Dako), MSH6 (44, 1:100, Bio SB, USA), MSH2 (G-219-1192, 1:100, Bio SB, USA), MLH1 (ab 92312, 1:100, Abcam, UK), and PMS2 (ab 110638, 1:100, Abcam, UK). After washing, slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and signal was developed using DAB (Lab Vision™ UltraVision™ ONE Detection System). For all IHC parameters, each specimen was assessed for percentage of positively staining cells and the intensity assessment was performed as follows: 0 = negative, 1 = low positive, 2 = medium positive, and 3 = strong positive. The intensity of staining and the number of stained cells were considered for each case and the product was calculated to obtain the h-score. Positive controls used for immunohistochemistry were tissue specimens of colon cancer, and negative controls were evaluated by omission of primary antibody in the tissue specimens.

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).