Serum free protein block buffer

Deparaffinized muscle sections (4 µm thick) were washed in PBS and antigen retrieval was performed using antigen retrieval buffer (Dako, Santa Clara, CA, USA) in an autoclave. Slides were blocked in Serum-Free Protein Block buffer (Dako) for 1 h at room temperature. Primary Goat anti-mouse Ang1 (AF923, R&D Systems, Minneapolis, MN, USA), rabbit anti laminin (Z0097, Dako), rat anti-mouse PDGFβ (ab51876, Abcam, Cambridge, UK), HA binding peptide Neurocan-dsRed [10 (link)] (200 µg/mL), or lectin from Bandeiraea simplicifolia isolectin B4 (BS-1-TRITC, L5264, Sigma or BS-1-BIOTIN, L2140, Sigma, Zwijndrecht, the Netherlands) were incubated overnight at 4°C in blocking buffer, followed by an appropriate secondary antibodies for 1 h at room temperature. Human diabetic muscle sections (4 µm thick) of critical limb ischemia biopsies treated as above and incubated overnight with Ncan-dsRed (200 µg/mL) and Rabbit anti-Human CD31 antibody (ab28364, Abcam) at 4 °C, followed by an appropriate secondary antibody for 1 h in blocking buffer, washed with PBS. Tissue slides were embedded in Prolong^TM gold antifade mountant with DAPI (P36931, ThermoFisher, Bleiswijk, the Netherlands) and recorded using a 3D Histech Pannoramic MIDI Scanner (Sysmex, Budapest, Hungary). Quantification was performed using the public domain NIH ImageJ software (FIJI version 1.49m; http://rsb.info.nih.gov/ij).

The NSCLC and normal lung tissues were fixed, embedded, sectioned, and deparaffinized. Some of the deparaffinized sections were stained with H&E staining. IHC staining was performed using a Dako Envision System (Dako, USA) following the manufacturer’s protocol. Sections were blocked using serum-free protein block buffer (DAKO, CA, USA) for 30 min, after which they were incubated with anti-Id-1 (1:200, Santa Cruz, USA).

Tissues were fixed, embedded, sectioned, and removed. Nonspecific antibody binding sites in sections were blocked with serum-free protein block buffer (DAKO) for 30 min. The sections were then incubated with anti-SYVN1 antibody (1: 200, Abcam).

To analyze the expression and localization of LHX8 and Lin-28 in rat ovaries following the transplantation of PD-MSCs, ovary samples were embedded in OCT compound (Fisher Scientific, Pittsburgh, PA, USA). Five-micron-thick cryostat sections were fixed in cold methanol for 10 min and permeabilized with proteinase K (Dako) for 5 min at RT. The sections were blocked with protein block serum-free buffer (Dako) at RT for 30 min and incubated first with goat anti-LHX8 (1:100 dilution, Santa Cruz) at 4 °C overnight and then with rabbit anti-Lin-28 (1:100 dilution, Abcam) in the dark at RT for 2 h. The mixture of secondary antibodies, including Alexa Fluor 488 chicken anti-goat IgG (1:200 dilution, Invitrogen) and Alexa Fluor 594 goat anti-rabbit IgG (1:200 dilution, Invitrogen), was incubated in antibody diluent (Dako) at RT for 1 h, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). The stained coverslips were mounted using a mounting solution to avoid light loss. Images were visualized using an Olympus confocal microscope (× 100 magnification) (Olympus, Tokyo, Japan; https://www.olympus-global.com).