Improved citrate antigen retrieval solution

Tissue slides were deparaffinized, covered with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, and then immersed in Improved Citrate Antigen Retrieval Solution (Beyotime) at sub-boiling temperature for 10 min. After blocking in 10% goat serum (Beyotime) with 0.2% Triton X-100 (Beyotime) in PBS for 1 h at room temperature, the slides were incubated with anti-GALNS antibody (1:200; Proteintech) at 4 °C in a humidified chamber. Subsequently, the sections were incubated with the GTVision III Detection System/Mo&Rb Kit (Gene Tech). Each specimen was scored according to the intensity of staining (0, none; 1, weak; 2, moderate; 3, strong) and the proportion of stained cells (0, 0%; 1, 1–24%; 2, 25–49%; 3, 50–74%; 4, 75–100%) according to the German semi-quantitative scoring system. The final immunoreactivity score was determined by multiplying the intensity with the positivity rate, and ranged from 0 to 12.

Tissues were fixed with 4% PFA, embedded in paraffin, and sectioned. H&E (G1005, Servicebio, China) and Masson's trichrome (G1006, Servicebio) staining were conducted according to the manufacturer's instructions, respectively. For immunofluorescence analysis, deparaffinized slices were incubated with an Improved Citrate Antigen Retrieval Solution (P0083, Beyotime), and stained with primary antibodies against Sftpc (ab211326, Abcam, USA), Ki67 (GB121141, Servicebio), β‐catenin (8480S, Cell Signaling Technology, USA), and DAPI (C1002, Beyotime). Tunel staining (G1502, Servicebio) was performed according to the manufacturer's instructions. For immunohistochemistry staining of macrophages and neutrophils, deparaffinized slices were incubated with F4/80 (GB113373, Servicebio) and Ly6G (GB11229, Servicebio) antibodies, respectively. For immunocytochemistry analysis, cells were fixed with 4% PFA and stained with primary antibodies against BRACHYURY (ab209665, Abcam) and Oct4 (sc‐5279, Santa Cruz).

Thymus tissue was fixed in 4% paraformaldehyde (biosharp life science, BL539A), embedded in paraffin, and sectioned to 4 μm thickness. Antigen retrieval was performed by boiling sections in Improved Citrate Antigen Retrieval Solution (Beyotime, P0083). Sections were blocked for 1 hour at room temperature using 5% BSA (Solarbio life sciences, SW3015), followed by incubation with IGFBP5 antibody (Abclonal, A12451) overnight at 4 °C. Staining with HRP Donkey Anti-Rabbit IgG (H+L) (Abclonal, AS038) was performed for 1 h at room temperature. Slides were developed using DAB (Beyotime, P0202) and counterstained with hematoxylin (Beyotime, C0107). All slides were scanned by PreciPoint M8 and analyzed with ViewPoint BETA v 0.8.2.7.

Adult SD rats were anesthetized by subcutaneous injection of pentobarbital sodium and fixed by transcardial perfusion with 4% paraformaldehyde in PBS containing 77.4 mM Na₂HPO₄, 22.6 mM NaH₂PO₄, pH 7.4. Small intestine was collected and stored in PBS supplemented with 0.1% PFA and 0.02% NaN₃. A cryo-section of 5 μm was baked at 60°C overnight, rehydrated in 1 × TBS (1 mM Tris, 150 mM NaCl, pH 7.4) at RT for 1 hr, followed by 5 washes with 1 × TBS. The section was then incubated at 98°C for 20 min in Improved Citrate Antigen Retrieval Solution (catalog no. P0083; Beyotime). After five washes with 1 × TBS, the section was blocked for 2 h with 5% normal goat serum in 1 × TBS, and then incubated with primary antibody at 4°C overnight. The section was washed 5 min × 5 times with 1 × TBS and incubated with DyLight-conjugated secondary antibody at RT for 1 h. After three washes with 1 × TBS, the section was counterstained with 4′,6-Diamidino-2-Phenylindole (DAPI) at room temperature for 5 min, washed three times with 1 × TBS, and mounted with Antifade Polyvinyl Pyrrolidone Medium (cat#P0123; Beyotime). Images were acquired on a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan).

Adult rats were fixed by transcardial perfusion with 4% paraformaldehyde (PFA) in PBS (in mM: 77.4 Na₂HPO₄, 22.6 NaH₂PO₄, pH 7.4). For immunofluorescence, cryo-sections of kidney were cut with thickness of 8 µm. The section was dehydrated overnight at 60°C and then rehydrated for 1 h in 1 × TBS (1 mM Tris, 150 mM NaCl, pH 7.4). After five 5-min washes with 1 × TBS, the section was incubated in Improved Citrate Antigen Retrieval Solution (catalogue no. P0083; Beyotime) at 98°C for 20 min. After five 5-min washes with 1 × TBS, the section was then blocked for 1 h with Immunol Staining Blocking Buffer (catalogue no. P0102; Beyotime) at RT, and incubated with primary antibody at 4°C overnight. The section was washed five times with 1 × TBS, incubated with DyLight-conjugated secondary antibody at room temperature for 1 h followed by five washes with 1 × TBS, and then mounted with Antifade Polyvinyl Pyrrolidone Medium (catalogue no. P0131; Beyotime). The mounting medium contains 4,6-Diamidino-2-Phenylindole (DAPI). Images were acquired on a FluoView FV3000 confocal microscope (Olympus, Tokyo, Japan).

The testicular sections were prepared as described above for HE staining. Then antigen retrieval using the Improved Citrate Antigen Retrieval Solution (P0083; Beyotime Biotechnology, Beijing, China) was performed at 100 °C for 10 min. Subsequently, the samples were cooled and washed three times with 0.3% Triton X-100 in PBS for 10 min, followed by three PBS washes. Afterward, the samples were blocked with 1% BSA for 30 min and then incubated with primary antibodies (Table 2) in 1% BSA overnight at 4 °C. Subsequently, the samples were washed three times with PBS and then incubated with the secondary antibody for 1 h at room temperature. Next, the samples were washed three times with PBS, and DAPI (D9542) was used to stain the cell nuclei for 10 min at room temperature. Finally, the slides were mounted in glycerol (ZEISS, Oberkochen, Germany) and imaged using a ZEISS Axiocam 503 mono fluorescence microscope.