Ab110638

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.

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).

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).

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.