Ab16660

Standard IHC protocol was followed to stain the tumor tissue samples using the mouse monoclonal antibody against hNIS (human Sodium Iodide Symporter) (Abcam, ab17795), ER (Estrogen Receptor) (Abcam, ab16660, ab288). Briefly, 5 µm sized paraffin embedded tissue sections were de-paraffinized with xylene and endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol for 30 minutes in the dark. Tissue sections were dehydrated through graded alcohols and subjected to antigen retrieval using 10mM sodium citrate. Sections were washed with TBST (Tris Borate Saline Tween-20) and then blocked with 5% BSA (Bovine Serum Albumin) for one hour. Slides were incubated with the respective mouse monoclonal primary antibody diluted with TBS. Slides were then washed for 5 minutes in TBST and incubated for 1 hour with the respective HRP (Horse Raddish Peroxidase) conjugated anti-mouse secondary antibody diluted with TBS in a ratio of 1∶200. After washing, slides were incubated with DAB (3,3′-diaminobenzidine tetrahydrochloride) (Sigma) and immediately washed under tap water after color development. Slides were then counter stained with hematoxylin. Slides were mounted with DPX (dibutyl phthalate xylene) and were then observed under a light microscope (Carl Zeiss).

Tissues and organoids were fixed with 4% paraformaldehyde, then dried, and paraffin-embedded. Sections were cut for HE staining and immunohistochemistry (IHC). Primary antibodies utilized in this work comprised anti-ER (Abcam, ab16660, 1 : 300), anti-PR (Abcam, ab101688, 1 : 300), and anti-HER2 (Abcam, ab16662, 1 : 100). All images were obtained using an Olympus microscope (Olympus, Japan).

Tissues were fixed in 4% paraformaldehyde and embedded in paraffin using standard protocols. For processing organoids, they were resuspended in cold PBS by pipetting, spun down, and fixed in 4% paraformaldehyde for 30 min. The fixed organoids were stained with haematoxylin before transfer to dehydration and paraffin embedding. Paraffin sections with a thickness of 5 µm were used for all analyses, H&E staining was carried out for histopathological evaluation. Immunohistochemistry was performed using the ELF 97 Immunohistochemistry Kit (ThermoFisher, E6600). Primary antibodies used in this study included Ki‐67 (CST, 9449S, 1:500), anti‐ER (Abcam, ab16660, 1:200), anti‐PR (Abcam, ab101688, 1:400), and anti‐HER2 (CST, 2165, 1:200). All images were captured on a Nikon microscope (Nikon, ECLIPSE, Ci).

Anti-estrogen receptor alpha antibody (ab16660) was purchased from Abcam (330 Cambridge Science Park, Cambridge, CB4 0FL, UK). Anti-mouse IgG HRP-linked antibody (7076S) and anti-rabbit IgG HRP-linked antibody (7074S) were purchased from Cell Signalling Technology (Hamilton House, Mabledon Place, London, WC1H 9BB, UK). Sodium tetrachloroaurate dihydrate, (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (EDC), N-hydroxysulfosuccinimide sodium salt (NHS), poly(ethylene glycol) 2-mercaptoethyl ether acetic acid (HS-PEG5000-COOH), dynasore hydrate, 1,2-bis(4-pyridyl)ethylene (BPE), 4-(2-hydroxyethyl)-1-piperazineethancesulfonic acid (HEPES), and 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Sigma-Aldrich Ltd (The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT, UK). LIVE/DEAD Viability/Cytotoxicity Assay Kit was purchased from ThermoFisher Scientific (3 Fountain Dr, Inchinnan, Renfrew PA4 9RF, UK). All glassware was cleaned in aqua regia (3 HCl : 1 HNO₃).

The primary antibodies used were anti-CK7 (ab181598, Abcam), anti-CK19 (ab52625, Abcam), anti-Ki67 (ab16667, Abcam), anti-vascular endothelial growth factor (VEGF) (ab52917, Abcam), anti-estrogen receptor (ERα) (ab16660, Abcam), and anti-progesterone receptor (PR) (ab16661, Abcam). The paraffin sections were deparaffinized and hydrated. The antigen was retrieved with EDTA buffer. The nonspecific antibody binding sites were blocked by goat serum. The sections were incubated at 4 °C overnight with primary antibodies and subsequently incubated with biotin-labeled secondary antibody at 37 °C for 1 h. The color reaction was developed with diaminobenzidine (DAB) (ZSGB Bio) and nuclei were stained with hematoxylin (Solarbio). The positive staining cells were observed as brownish-yellow under the microscope. Image J was applied to measure the percentage of positive staining area.

Anti-estrogen receptor alpha (ERα)
antibody (ab16660) and Anti-Erb2 (HER2) antibody (ab16899) were purchased
from Abcam (330 Cambridge Science Park, Cambridge, CB4 0FL, UK). Anti-mouse
IgG HRP-linked antibody (7076S) and anti-rabbit IgG HRP-linked antibody
(7074S) were purchased from Cell Signalling Technology (Hamilton House,
Mabledon Place, London, WC1H 9BB, UK). Sodium tetrachloroaurate dihydrate,
(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride) (EDC), N-hydroxysulfosuccinimide sodium
salt (NHS), poly(ethylene glycol) 2-mercaptoethyl ether acetic acid
(HS-PEG5000-COOH), dynasore hydrate, 1,2-bis(4-pyridyl) ethylene (BPE),
4-(2-hydroxyethyl)-1-piperazineethancesulfonic acid (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES), LIVE/DEAD Viability/Cytotoxicity
Assay Kit diacetate (FDA)-propidium iodide (PI), and Synperonic F108
were obtained from Sigma-Aldrich Ltd. (The Old Brickyard, New Road,
Gillingham, Dorset, SP8 4XT, UK). The LIVE/DEAD Viability/Cytotoxicity
Assay Kit was purchased from ThermoFisher Scientific (3 Fountain Drive,
Inchinnan, Renfrew PA4 9RF, UK). Milli-Q deionized water was used
after purification using a Milli-Q purification system. All glassware
was cleaned in aqua regia (3 HCl:1 HNO₃).