Eclipse e600 microscope

Tissues and organoids were fixed in 4% paraformaldehyde, followed by dehydration, paraffin embedding, sectioning, and standard hematoxylin and eosin (H&E) staining and IHC staining. Next, the samples were incubated with primary antibodies, including anti-thyroid transcription factor-1 (TTF-1) (1:200 dilution; Invitrogen Signaling Technology), anti-p40 (1:200 dilution; Cell Signaling Technology), anti-cytokeratin 5 and 6 (1:200 dilution, #MA5-12429; Invitrogen), and anti-programmed death ligand-1 (anti-PD-L1) (1:200 dilution, #13684; Cell Signaling Technology). The sections were subsequently incubated with secondary antibodies (#AI-2000 and #AI-1000; Vector Laboratories, Newark, CA, USA) at 1:5000 dilution and visualized using an ultraView Universal DAB Detection kit (Ventana Medical Systems, Oro Valley, AZ, USA). Nuclei were counterstained with Harris hematoxylin. Images were acquired via a Leica Eclipse E600 microscope [16 (link)].

Tissues and organoids were fixed in 4% paraformaldehyde (PFA) followed by dehydration, paraffin embedding, sectioning, and standard H&E staining. For IHC staining, the samples were incubated with primary antibodies including anti-napsin A (1:200 dilutions; #NCL-L-Napsin A, Novocastra, IL, USA), anti-TTF-1 (1:200 dilutions; #NCL-L-TTF-1, Novocastra), anti-CK7 (1:400 dilutions; #M7018, Dako, CA, USA), anti-p63 (1:200 dilutions; #M731701-2, Dako), anti-cytokeratin 5/6 (CK5/6; 1:200 dilutions; # MA5-12429, Invitrogen), anti-CD133 (1:200 dilutions; #64326, Cell Signaling Technology, MA, USA), anti-PD-L1 (1:200 dilutions; #13684, Cell Signaling Technology), and anti-c-Met (1:400 dilutions; #257261, Dako). The sections were subsequently incubated with secondary antibodies (#AI-2000, #AI-1000, Vector laboratories, CA, USA) for 1:5000 dilutions and visualised using the ultraView Universal DAB Detection kit (Ventana Medical Systems, AZ, USA). Nuclei were counterstained with Harris haematoxylin. Images were acquired on a Leica Eclipse E600 microscope.

After fixed with 4% paraformaldehyde, Matrigel-organoids was aspirated from the 24-well plates into a 1.5 mL EP tube and centrifuged at 1000 rpm for 5 min, then embedded in 2% agarose. The solidified organoids-agarose was fixed in 4% paraformaldehyde overnight, and followed by gradient dehydration the next day, treated with dimethylbenzene for 40 min before embedded in paraffin. 4 μm paraffin sections were cleared with dimethylbenzene and rehydrated with gradient ethanol. For Immunohistochemistry (IHC) assay, the paraffin section antigen retrieval was performed by microwave heating and stained with the following antibodies: Rabbit anti-ER antibody (Santa, Dallas, Texas, USA, sc-8002, 1:300), anti-PR (CST, Danvers, MA, USA, 8757s, 1:600), anti-HER2 (CST, 2165s, 1:600), and anti-Ki67 (CST, 9449s, 1:800). DAB kit was purchased from Zhongshan Goldenbridge Biotechnology Company (Beijing, China). Nuclei were counterstained with haematoxylin. Images were acquired with Leica Eclipse E600 microscope.