Antibody diluent reagent solution

Isolated tissues were fixed in fresh 4% paraformaldehyde, embedded in paraffin, and cut into 5-6 µm sections using a Leica RM2155 microtome and Super Plus charged slides. Cerebellum (CB) slices were sectioned into sagittal sections, and hippocampus (HC) sections were in coronal orientation. For IHC, the primary antibody was used at optimal ratio in Antibody Diluent Reagent Solution (Life Technologies) and incubated overnight at 4°. Slides were then washed and incubated with secondary antibody (1:200) in antibody diluent for 1 hr at room temperature. Slides were further washed and stained with Hoecsht 3342 and scanned under Zeiss LSM 710 Confocal Microscope for further analysis. Primary antibodies: MAP2 (Abcam ab5392, 1:300), AUTS2 (Sigma HPA000390, 1:200), WBSCR17 (LSBio LS-B14501, 1:250), Calbindin D-28K (Sigma C9848, 1:300), Calretinin (Santa Cruz sc-365989, 1:300), CNPase (Millipore MAB326, 1:250), DRD2 (Millipore AB5084P, 1:200), GFAP (Invitrogen 180063, 1:300), GAD 65/67 (Santa Cruz sc-365180, 1:200), Tbr2 (Abcam ab23345, 1:100), cFos (Abcam ab190298, 1:200); Secondary antibodies (Thermo-Fisher Scientific): Goat anti-Mouse IgG (Alexa Fluor 488, A11001), Goat anti-Rabbit IgG (Alexa Fluor 594, A11012), Donkey anti-Rat IgG (Alexa Fluor 594, A21209).

GH3, A7, and M2 cells were seeded on 13-mm poly-L-lysine-coated coverslips at a density of 1.25 × 10⁵ cells/well in 24-well plates and grown at 37°C for 18 h. The following day, cells were incubated with pasireotide 100 nM or octreotide 100 nM for 0, 5, or 30 min. For immunofluorescence analysis of SST₂ and SST₅ localization in GH3, A7, and M2 cells, rabbit anti-SST₂ UMB1 #ab134152 (1:50, Abcam, Cambridge, UK) and mouse anti-SST₅ #6675-1-Ig (1:200, Proteintech, Rosemont, IL, USA) antibodies were used and incubated o/n at 4°C. Anti-mouse Alexa Fluor™ 546-conjugated secondary antibody (1:500, Thermo Fisher Scientific, CA, USA) and anti-rabbit Alexa Fluor™ 488-conjugated secondary antibody (1:500, Thermo Fisher Scientific, CA, USA) were incubated at room temperature for 2 h. All antibodies were diluted in Antibody Diluent Reagent Solution (Life Technologies, Thermo Fisher, CA, USA). Coverslips were mounted on glass slides with EverBrite™ Hardset Mounting Medium with DAPI (Biotium, Fremont, CA, USA) for subsequent observation under epifluorescence microscope. The NIH ImageJ software was used to merge single-channel images before and after deconvolution.

Paraffin blocks were cut into slices at a thickness of 2 μm, which were collected on polylysine-coated glass slides. Endogenous peroxidase activity was blocked with 3% H₂O₂, and antigens were retrieved with universal antigen retrieval solution for immunohistochemistry (Typing, China). The slices were incubated with primary antibodies, which were diluted in antibody diluent reagent solution (Life Technologies, USA), at 4 °C overnight. The sections were incubated with HRP-conjugated secondary antibodies, exposed to DAB to visualize the antigen signals, and counterstained with hematoxylin. After the sections were sealed with a neutral resin, the positive reactants were viewed under a microscope (DM6 B, Leica, Germany). Immunohistochemical staining was quantified by mean optical density using Image-Pro Plus, and five random fields per sample were used for counting. The antibodies used in this study are listed in Supplementary Table S2.

Histones extracted from PBMCs were assessed for protein concentration using a Thermo Scientific™ Pierce™ BCA Protein Assay Kit (Cat#23227). Based on the protein concentration results, 15 μg total protein was combined with 5 μl 5× loading buffer and 2% SDS to a final volume of 20 μl and separated on a 5% laminated glue + 15% separation gel at 80 V/gel for stacking and 120 V/gel for resolution. Wet transfer to a 0.2-μm PVDF membrane (Immobilon™-P^SQ membrane) was performed at 200 mA for 3 hours. The membranes were soaked in blocking buffer (5% skimmed milk) for 2 hours. Primary antibodies were applied overnight at 4°C [anti-lactyl-histone H3 (Lys18) rabbit mAb (PTM-1406RM; Lot: K111421; 1:1000 dilution in Life Technologies™ Antibody Diluent Reagent Solution cat# 003218) and anti-histone H3 antibody Nuclear Marker and ChIP Grade (ab1791) (1:1000 dilution in Life Technologies™ Antibody Diluent Reagent Solution)]. The secondary antibody [goat anti-rabbit IgG H&L (HRP) (ab6721)] diluted 1:3000 in TBS-T with 5% milk was added and incubated at room temperature for 2 hours. The bands detected were quantitatively analysed using VILBER Fusion Solo S.

caspase-3, e-NOS, and TNFR activity was investigated in corneal sections using immunohistochemical methods. The primary antibodies were caspase-3 (Bioss, Woburn, MA, USA; Cat. No:bs-0081R), e-NOS (Bioss, Cat. No:bs-0163R), TNFR1 (Bioss, Cat. No:bs-2941R). All primary antibodies were diluted with 1:100 Antibody Diluent Reagent Solution (Life Technologies, Carlsbad, CA, USA; Cat. No: 003118). The block solution, streptavidin peroxidase solution, and secondary antibody were used as kits (Cat. No: TP-125-HL). Sections that were 4–5 μm thick were deparaffinized and dehydrated. The sections were then incubated with 3% hydrogen peroxide, Ultra-V Block (ThermoFisher, Waltham, MA, USA), primary antibodies (Bioss), secondary antibody (ThermoFisher), and streptavidin peroxidase (ThermoFisher). Marking with DAB solution (Vectorlab, Burlingame, CA, USA) and nuclear staining with hematoxylin were performed. In this study, a previously reported semiquantitative scoring system was used to evaluate immunoreactivity [20 (link)]. Immunostaining intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentages of positively stained cells were calculated and scored as follows: 0–4% = 1, 5–19% = 2, 20–39% = 3, 40–59% = 4, 60–79% = 5, and 80–100% = 6. Finally, multiplicative quick scores were calculated [21 (link)].

Whole-cell lysates were prepared with lysis buffer (1% SDS, 1% protease inhibitors, 3 μM TSA, 50 mM NAM) and sonication. The lysis mixture was centrifuged at 12000 x g for 10 minutes at 4°C to remove cell debris, and the supernatant was transferred to new tubes. Protein concentrations were determined using a Thermo Scientific™ Pierce™ BCA Protein Assay Kit (Cat#23227). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Based on protein concentration results, 15 μg total protein was mixed with 5 μl 4× loading buffer and 2% SDS to a final volume of 20 μl and separated on an SDS-12% polyacrylamide gel at 15 mA/gel for approximately 15 minutes for stacking and at 35 mA/gel for resolution. Staining was performed using Coomassie Blue (R-250) for 2 hours at room temperature, and the gel was then decolorized. For western blotting, anti-lactyl-lysine antibody (PTM-1401RM; Lot: K111421; 1:1000 dilution in Life Technologies™ Antibody Diluent Reagent Solution cat# 003218) was used as the primary antibody and incubated overnight at 4°C. The secondary antibody was goat anti-rabbit IgG (H+L) (Thermo Pierce, Peroxidase Conjugated, 31460) diluted 1:10,000 in TBS-T with 5% milk at room temperature for 2 hours. Bands were detected quantitatively using VILBER Fusion Solo S.