Tris buffered saline tween 20

All tissue specimens were fixed for 16–24 hours in 10% neutral buffered formalin (NBF) and embedded in paraffin. Immunohistochemistry (IHC) was performed on tumor tissue sections (5 μm thick) with anti-human antibodies p-AKT(S473) and p-PRAS40(T246). After deparaffinization and rehydration of the tissue sections, antigen retrieval was conducted in a decloaking chamber for 30 minutes at 95°C, followed by 10 seconds at 90°C using citrate buffer (pH 6, Poly Scientific, Bay Shore, NY). IHC was performed using a Labvision 480 autostainer (Thermo Fisher Scientific). Rabbit on Rodent HRP-Polymer (Biocare Medical, Concord, CA) was used as a secondary antibody. Primary antibodies were diluted in 1% BSA in Tris-buffered Saline-Tween 20 (Sigma Aldrich). Tissue sections were incubated with primary antibody for 30 minutes and then with polymer for 25 minutes at ambient temperature. The reagent 3,3’-diaminobenzidine (DAB) was used as the chromogen and the slides were counterstained with hematoxylin after IHC. Images were captured using an Aperio Scanscope (Aperio, Vista, CA).

After fixation, tissues were embedded in paraffin, sectioned, and stained for immunohistochemical analysis. Antigen unmasking was performed by treatment with sodium citrate buffer at pH 6, and tissue sections were stained for R. australis using rabbit polyclonal Abs against rickettsiae and ATG5 (catalog number NB110-53818; Novus). Biotinylated secondary Abs (Vector Labs) were diluted in Dako Ab diluent (catalog number S3022; Dako). The tertiary reagents and streptavidin-alkaline phosphatase were diluted in the same Dako diluent, as were the primary Abs (73 (link)). All washes were with Tris-buffered saline–Tween 20 (Sigma). Sections were dehydrated before synthetic glass coverslips were mounted with Permount mounting medium. All the sections were photographed with an Olympus DP71 camera (Olympus, Center Valley, PA, USA) attached to an Olympus Ix71 inverted microscope (Olympus, Tokyo, Japan) utilizing ×20 objectives.

RPMI 1640 medium, gentamicin, trypsin, phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were acquired from Gibco (Invitrogen, Carlsbad, CA, USA). Isolated compounds 7-hydroxy-3,4-dihydrocadalene (>98.0%), 7-(phenylcarbamate)-3,4-dihydrocadalene (77% yield) and 7-(phenylcarbamate)-cadalene (79% yield) were generously provided by Dr. Guillermo Delgado Lamas (Instituto de Química, UNAM). Their isolation or preparation and structural characterization are described by Egas et al. (2017) (link).
Dimethyl Sulphoxide (DMSO), H₂O₂, 2′,7′-Dichlorofluorescein Diacetate (DCFH-DA), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Bovine Serum Albumin (BSA), 2-thiobarbituric acid, antimycin A, oligomycin, rotenone, RIPA buffer, phosphatase inhibitor cocktails, bicinchoninic acid reagent, Tris Buffered Saline-Tween 20, GAPDH antibody (G-8795), caspase 3 (sc-7148) and Bcl-2 (sc-492) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail from Roche (Lewes, UK). Polyvinylidene difluoride (PVDF) membranes from GE Healthcare (Buckinghamshire, UK).

Kidney tissues were lysed in radioimmunoprecipitation (Beyotime) assay lysis buffer containing phenylmethylsulfonyl fluoride (Solarbio). Protein concentrations were determined using a bicinchoninic acid assay. Protein samples were separated on a 30% sodium dodecyl sulfate–polyacrylamide gel at 120 V and then transferred to nitrocellulose membranes (Pall Corporation) using a Turbo Transfer System (Bio‐Rad). Membranes were blocked for 1 h at 25 °C and then incubated with the appropriate primary antibodies at 4°C overnight, followed by washing three times with Tris‐buffered saline‐Tween 20 (Sigma Aldrich). Membranes were then incubated with horseradish peroxidase‐conjugated secondary antibodies for 2 h at 25°C and washed with Tris‐buffered saline‐Tween 20. Protein bands were visualized using chemiluminescence with ECL Plus (#P1050; Applygen) according to the manufacturer's instructions. The primary antibodies used in our study were as follows: TGF‐β1 (1:1000, #21898; Proteintech), α‐SMA (1:1000, #55135; Proteintech), Smad3 (1:1000, #ab52903; Abcam), p‐Smad3 (1:1000, ab40854; Abcam), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 1:1000, #10494‐1‐AP; Proteintech).

Western blotting was performed according to a previous method [36 (link)]. Protein isolation from mouse ovaries and GCs was performed using the RIPA lysis buffer (R0020, Solarbio). A BCA protein assay kit (PC0020, Solarbio) was used to measure the protein concentration. The protein samples were denatured at 100°C for 10 min. Then, the proteins were separated by sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes subsequently were blocked using 5% skimmed milk for 1 h at room temperature. After blocking, the membranes were incubated overnight with primary antibodies against CD63 (ab134045, 1 : 1000; Abcam), calnexin (ab133615, 1 : 1000; Abcam), HBP1 (ab216844, 1 : 1000; Abcam), T cell factor (TCF; ab272235, 1 : 1000; Abcam), lymphoid enhancer factor (LEF; ab137872, 1 : 1000; Abcam), p-GSK3β (ab68476, 1 : 1000; Abcam), histone H3 (ab1791, 1 : 1000; Abcam), GSK3β (ab185141, 1 : 5000; Abcam), β-catenin (ab184919, 1 : 1000; Abcam), and GAPDH (ab9485, 1 : 2500; Abcam). The next day, the membranes were washed three times using Tris-buffered saline Tween-20 (Sigma-Aldrich) and incubated with secondary antibodies at room temperature for 1 h. The protein level was detected using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).