Ki67 clone sp6

Monoclonal antibodies: CD44 clone IM7 (Biolegend, Ozyme, Saint Quentin Yvelines, France; used at 1/200); Ki67 clone SP6 (Abcam, Paris, France; used at 1/500); GSK3β clone 7 (BD Transduction Laboratories; used at 1/2000). Polyclonal antibodies: PAR₂ antibody was from Santa Cruz Biotechnology (Dallas, TX, USA; used at 1/100); P(Ser21/9)GSK3 (Cell Signaling Technology, Ozyme, Saint Quentin Yvelines, France; used at 1/50 for immunofluorescence and 1/1000 for Western blot); Alexa Fluor 488- and Alexa Fluor 555-conjugated secondary antibodies (Invitrogen Molecular Probes, Thermo Fisher Scientific, Illkirch, France; used at 1/1000). Pharmacological inhibitors: GSK3 inhibitor SB-216763 was from Tocris Bioscience (RD Systems, Lille, France); Rho kinase inhibitor Y-27632 was from Sigma (Saint-Quentin Fallavier, France).

To gauge severity of hepatic injury, haematoxylin and eosin (H&E)‐stained sections were graded as follows: (0) no or minimal injury; (1) mild injury (i.e. cytoplasmic vacuolation and focal nuclear pyknosis); (2) moderate or severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia and loss of intercellular bridges; (3) severe necrosis, marked by hepatic cord disintegration, haemorrhage and neutrophilic infiltrates; and (4) very severe necrosis, showing the latter manifestations to extreme degree 25. Hepatic steatosis was assessed via Oil Red O staining of frozen sections, with percentages rendered from standard image analysis 25. Hepatic samples analysed for regeneration were immunostained using rabbit monoclonal antibody to Ki 67 (clone SP6; Abcam, Cambridge, UK), diaminobenzidine (for colorization) and haematoxylin counterstain 25. At least 30 high‐power fields were counted per slide.

The following antibodies were used for IHC and Western immunoblotting: ATF4 (catalog 10835-1-AP) and SKP2 (catalog 15010-1-AP) from Proteintech; CHOP (catalog 2895), p-YAP (catalog 13008, S127), YAP (catalog 14074), p27 (catalog 3686), MYC (catalog 9402), p-RPS6 (catalog 4858, S235/236), t-RPS6 (catalog 2217), p-mTORC1 (catalog 5536, S2448), t-mTORC1 (catalog 2983), p-ERK1/2 (catalog 9101, T202/Y204), t-eIF2α (catalog 2103), p-AKT (catalog 9271, S473), t-AKT (catalog 4691), EIF4FBP1 (catalog 2855, T37/46), K48-Ub (catalog 8081), p-PERK (catalog 3179, T980), and PERK (catalog 3192) from Cell Signaling Technology; CYR61 (catalog E-AB-14920), CTGF (E-AB-12339), and aquaporin 2 (E-AB-30540) from Elabscience Biotechnology; CDK1 (catalog SC-54) and t-ERK1/2 (catalog SC-94) from Santa Cruz Biotechnology; α-tubulin (catalog T5168) and K63-Ub (catalog 05-1308) from MilliporeSigma; PCNA (catalog MS-106-P1ABX and, p-LATS1/2 (catalog PA5-64591, S809/S872) from Thermo Fisher Scientific; Oct4 (catalog NB100-2379) from Novus Biologicals; Sox2 (catalog ab97959), vimentin (catalog ab92547), Ki-67 (clone SP6), p-eIF2α (catalog ab32157, S51), BIM (catalog ab32158), and TAZ (catalog ab224239) from Abcam; GRP78 (catalog A0241) from StressMarq Biosciences; and nestin 1 (monoclonal rat-401s) from DSHBU Iowa.

Rats were euthanized with an intraperitoneal injection of a lethal dose of ketamine and xylazine and intracardially perfused with 0.1 M of PBS for 5 min. Afterward, their brains were removed and fixed in 10% buffered formalin, with wiring material in alcohols, pouring into paraffin. Paraffin sections were stained with hematoxylin and eosin, and IHC studies were performed using the REVEAL Polyvalent HRP-DAB Detection System. Monoclonal antibody Abcam (USA): Bcl-2/bax (MAB8272), LC3b (ab192890), Ki67 (clone SP6, ab16667), clathrin (clone EPR24231-72, ab271185), caveolin (clone E249, ab32577), Fas (ab216636), p53 (ab131442), and LC3b (ab192890) were used at a dilution of 1:100 to the antibody. When staining with ICH markers, positive and negative controls were used to exclude false-negative and false-positive results, to create standardization of the staining conditions and increase the objectivity of the results. The percentage of positively expressing cells in 10 fields of view for each sample and the intensity of immunohistochemical reactions (weak, moderate and pronounced) were calculated. All studies were performed using a MicroVisor of medical transmitted light μVizo-103 (LOMO, St Petersburg, Russia) with a magnification of 774.

The slides containing the prepared brain tissue sections were dewaxed and then rehydrated for 10 min. After that, the endogenous peroxidase activity was blocked using 3% hydrogen peroxide. The Ki-67 epitope was recovered by using the heat-induced epitope retrieval method in citrate buffer (pH = 6) with incubation for three minutes in a microwave oven. Then, the slides were incubated for two hours with primary antibodies Ki-67-clone SP6 obtained from Abcam, Waltham, MA, USA (Catalog number ab16667) followed by application of biotinized secondary antibodies (Biorbyt Ltd., Cambridge, UK, catalog number orb389335). The immunoreactivity for Ki-67 was visualized by utilizing extravidin peroxidase and 3,3′-diaminobenzidine as chromogen purchased from Sigma-Aldrich Co., St. Louis, MO, USA. The immunoexpression of Ki-67 was detected under a light microscope (Olympus, Japan) and was graded as follows: (+) denotes mild immunoexpression of Ki-67; (++) denotes moderate immunoexpression of Ki-67; and (+++) refers to massive immunoexpression of Ki-67 in the brain tissues [65 (link)].