Hematoxylin

A 10% neutral buffered formalin solution was used to fix the testis for at least one week prior to HE staining. Approximately 3 mm thick testicular tissues were dehydrated, rendered transparent, and boiled in wax before being embedded in paraffin. The sections (3 μm) were dried for 48 h at 60 ° C and dewaxed in water before being dipped in hematoxylin (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China) for 15 min and washed in tap water for 5 min. Subsequently, the sections were dipped in eosin (Sinopharm Chemical Reagent Beijing Co., Ltd.) for 15 s, then washed again for 5 min. After dehydration of the alcohol gradient, the clearance with xylene and placement of a cover slip were performed. Observations were conducted using the LEICA DM6000 light microscope (Leica, Wetzlar, Germany).

Interphotoreceptor retinoid-binding protein (IRBP) peptide (amino acids 1177–1191; sequence, ADG SSW EGV GVV PDV) and primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Complete Freund's Adjuvant (CFA) and 2-mercaptoethanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mycobacterium tuberculosis (TB, strain H37RA) was purchased from Difco (Difco Laboratories, Detroit, MI, USA). Recombinant rat IL-2, fluorescein isothiocyanate- (FITC-) conjugated TLR4 antibody, and phycoerythrin- (PE-) conjugated IL-10 antibody were purchased from Peprotech (Rocky Hill, NJ, USA), Thermo Fisher Scientific (Waltham, MA, USA), and BD Pharmingen (Mountain View, CA, USA), respectively. Phosphate buffer saline (PBS, pH 7.4, 20 mmol/L), formaldehyde, paraffin, hematoxylin, and eosin (HE) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RPMI 1640 medium was purchased from Gibco; Thermo Fisher Scientific, Ltd. (Waltham, MA, USA). IL-10 (JYM0651Ra) ELISA kit was purchased from Dakewe Biotech Co., Ltd. (Beijing, China). Toll-like receptor 4 (JYM0085Ra) ELISA kit was purchased from Wuhan ColorfulGene biological technology Co., Ltd. (Wuhan, China).

Briefly, tumor tissues embedded in paraffin were obtained from our previous experiments [26 (link)]. Samples were sectioned, stained with hematoxylin (Sinopharm Chemical Reagent Co., Ltd., Beijing, China) and antibodies against PCNA, p21, MMP-2, MMP-9, NFκB p65, CHOP, LC3B, vimentin, Snail or Slug. The primary antibodies were applied at 1:200 for PCNA, 1:200 for p21, 1:400 for MMP-2, 1:400 for MMP-9, 1:1000 for NFκB p65, 1:200 for CHOP, 1:400 for LC3B, 1:200 for vimentin, 1:200 for Snail and 1:200 for Slug. Finally, sections were mounted with DPX mounting media (Sigma-Aldrich), and images were obtained using a microscope (Leica, Wetzlar, Germany).

In order to evaluate the influence of MT exposure during the gonadal differentiation of S. schlegelii, first, the sex of each individual was determined through PCR. And, histological analysis was performed on S. schlegelii gonads at 140 dpb (n = 10–15/per group) for both the control group (XX and XY) and those subjected to MT treatments (XX) (20 ppm, 40 ppm, and 60 ppm). Briefly, the gonads were first collected and fixed overnight in 4% paraformaldehyde (Solarbio, Beijing, China). Posteriorly, the samples were dehydrated in alcohol (Sinopharm, Beijing, China) and xylene (BaSO) and embedded in paraffin wax (Leica, Wetzlar, LLH, Germany). Then, histological sections of 3 μm thickness were obtained and stained with hematoxylin (BaSO) and eosin (Sinopharm) for observation of general cellular structures under a microscope.

Collagen II immunofluorescence staining was performed as follows: the monolayer cells were dripped on the cover glass, fixed with 4% paraformaldehyde for 20 min and incubated with 3% H₂O₂ (Sinopharm, Beijing, China) at room temperature for 10 min. After that, the cells were rinsed with phosphate buffer saline (PBS) (Hyclone, Logan, UT, USA) for 5 min 3 times and incubated with 5% bovine serum albumin (BSA) (Sigma-Aldrich) for 10 min, and the serum was removed. The cells were added with rabbit Collagen II antibody (ab34712, 1:200, Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, the cells were rinsed thrice with PBS, incubated with biotin-labeled goat anti-rabbit IgG secondary antibody (1:100, A0277, Beyotime, Shanghai, China) for 10 min and rinsed with PBS 3 times. Finally, the cells were cultured with 2,4-diaminobutyric acid, re-stained with hematoxylin (Sinopharm), dehydrated with absolute ethanol and observed under an optical microscope.
Toluidine blue staining was performed as follows: the chondrocytes were seeded in 6-well plates with pre-fixed sterile cover glass. After adhesion, the cells were collected and fixed with 40 g/L polytoluene for 20 min and 75% ethanol for 15 min, and stained with 1% toluidine blue for 2–3 h. Then, the cells were rapidly rinsed with absolute ethanol, cleared with xylene (Sinopharm) and observed under an optical microscope.