Dab substrate kit

Immunohistochemical staining was performed according to the standard methods. Briefly, 5 µm sections from the tissue microarrays were baked at 70°C for 2 h. Sections were then deparaffinized in xylene, rehydrated using an ethanol concentration gradient, and heated in a pressure cooker for 3 min in an antigen retrieval citra solution (pH 8.0, catalog number ZLI-9072, Zhongshan Golden Bridge Biotechnology) for antigen repair, and sealed with 3% hydrogen peroxide for 15 min to inhibit the activity of endogenous peroxidase. Subsequently, tissue microarray slices were incubated for 16 h at 4°C with the primary antibody against DDX60L (dilution, 1:400; catalog number bs-14236R; Beijing Biosynthesis Biotechnology Co., Ltd.), followed by incubation with EnVision+ System-HRP (catalog number K4001, Dako). Finally, the staining was visualized with the DAB Substrate kit (catalog number DA1010-2, Solarbio) and counterstained with hematoxylin (22 (link)).

Cauda epididymis and testes from adult mice were fixed in Bouin's solution (Sigma, HT10132‐1L) at 4°C for 24 h, then embedded in paraffin, and cross‐sectioned. Sections mounted on glass slides were dewaxed and stained with haematoxylin and eosin (HE) for cauda epididymis, or with Periodic acid Schiff (PAS) for testes using PAS staining kit (KeyGEN BioTECH, KGA222). For in situ cell death analysis, testes from adult mice were fixed in 4% Paraformaldehyde Fix Solution (Beyotime, P0099), and TUNEL staining was performed using an In Situ Cell Death Detection Kit, POD (Roche, 11684817910) and DAB Substrate kit (Solarbio, DA1010).

Slides cut from paraffin-embedded tissues underwent drying, rehydration, antigen retrieval, and permeation before the samples were blocked in goat serum for 20 min and incubated in the primary antibody then in the second antibody. Color development was performed with a DAB Substrate kit (Solarbio, DA1010, Beijing, China) and counterstained with Hematoxylin (Solarbio, H8070, Beijing, China).

Twenty-four male and female ICR mice were weighed 20–22 g (Yisi experimental animal technology company, China). These mice were randomized into 3 groups (n = 6). All mice were fainted by injection 5% chloral hydrate solution. The hair of mice’s backside was shaved and depilated completely. The 40 µL of physiological saline buffer was applied on the back as a blank control group (Group A). The 20 µg EGF protein in 40 µL PBS solution was smeared as a positive control group (Group B). The 40 µL of the oil body expressed oleosin-hEGF-hEGF fusion protein was smeared as a sample group (Group C). The medication area was taken 1 cm² on mice’s backside. The skin tissues were collected after transdermal absorption for 30, 60 and 90 min duration in each group respectively. The collected tissues were embedded in which paraffin sections were created. Tissue slices were blocked and then incubated with the primary (rabbit Anti-hEGF, Bioss, bs-4568R, Lot: C-A1729, China) and secondary antibodies (Goat anti-Rabbit IgG ZSGB-BIO, ZLI-9019, Lot:K176904D, China). These tissue sections were stained with DAB substrate kit (Solarbio, China) and then hematoxylin (Solarbio, China) counterstain for 1 min. All photos were recorded using an Olympus confocal microscope (Olympus, Japan) [28 (link)].

H4, SW1783, and HMF cell lines were purchased from ATCC. According to the manufacturer’s instructions, total RNA was isolated using Trizol reagent (Invitrogen, USA). 2μg of the total RNA was transcribed into cDNA. SYBR Green PCR kit (Takara, Japan) was used for qRT-PCR. We selected the 2−ΔΔCq method to calculate gene transcription level, with β-actin mRNA as control. Data represent the mean ± SD of triplicate real-time PCR. The primers were synthesized by Tsingke Biotechnology (Shanghai, China) and displayed in Supplementary Table S1. Immunohistochemistry (IHC) analyzed the protein expression levels. GSAP (ab106630), LATS2 (ab111054), SWAP70 (ab228846), and SLC2A10 (ab110528) antibody was purchased from Abcam. EMP3 (sc-81797) antibody was purchased from Santa Cruz Biotechnology. Clinical characteristics of LGGs patients cohort are displayed in Supplementary Table S2. All the patients and the hospital’s Ethics Committee approved this research. LGGs tissues were formalin-fixed, paraffin-embedded, and sectioned at 4 µm. Immune complexes were detected with the SP Kit (Solarbio, Beijing, China) and DAB Substrate Kit (Solarbio, Beijing, China). Signals were detected using an Olympus BX41 microscope. Quantification of Immunohistochemistry (IHC) staining was performed in a blinded fashion.

Purified IgG1-Fc region and GalNAc-T1 proteins were treated with peptide N-glycosidase F (PNGase F, New England Biolabs), following the manufacturer’s protocol. Samples were run on 12% SDS-PAGE gels with or without DTT reduction, and transferred onto polyvinylidene fluoride membranes for 90 min. After blocked in 5% BSA or 1% polyvinylpyrrolidone (Sigma) the membranes were incubated with His-tag antibody or ConA-B respectively at 4 °C overnight. Blots were developed with DAB Substrate kit (Solarbio, China) following incubation with HRP-conjugated secondary antibody for 1 h at room temperature.