Ab198684

Human bladder tumor tissues were prepared into 5-μm section and subjected to immunohistochemical stain with Biotin-Streptavidin HRP.
Detection System (ZSGB-BIO) following the provider’s protocol. Siglec-15 antibody was obtained from Abcam (ab198684) and 1:50 dilution was applied. Diaminobenzidine method was adopted for signal development. The nuclei were counterstained with hematoxylin for reference purpose. Representative images were acquired using a DMi8 Leica DMi8 Inverted Microscope.

Multiplex immunofluorescence staining was obtained using PANO7-plex IHC kit, cat 0004100100 (Panovue, Beijing, China). Mouse anti-human Siglec15(Abcam, ab198684, 1:100), rabbit anti-human CD4(Abcam, ab133616, 1:500), mouse anti-human CD8A (CST, CST70306, 1:200), mouse anti-human FoxP3(Abcam, ab20034, 1:50), rabbit anti-human CD11b (CST, CST49420, 1:200), rabbit anti-human CD33 (Abcam, ab199432, 1:100), rabbit anti-human CD163(CST, CST93498, 1:500) were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification.
The Mantra System (PerkinElmer, Waltham, Massachusetts, US) were used to scan the stained slides to obtain multispectral images. the scans were combined to build a single stack image.
Single-stained and unstained sections of images were used to extract the spectrum of autofluorescence of tissues and each fluorescein, respectively, which were further used to establish a spectral library required for multispectral unmixing by inform image analysis software (PerkinElmer, Waltham, Massachusetts, US). We obtained reconstructed images of sections with the autofluorescence removed by using this spectral library. For each patient, a total of 8-15 high-power fields were taken based on their tumor sizes.

FFPE tissue sections (4-µM in thickness) was deparaffinized, and rehydrated. Subsequently, antigen-retrieval was performed in the waterbath. Primary antibodies included Anti-Siglec15 (1:35; ab198684, Abcam) and Anti-CD163 (1:200; clone number [EPR19518]; ab182422, Abcam) were diluted in PBS containing 1% BSA. Slides were incubated overnight at 4°C, and then washed and incubated with corresponding secondary antibodies (1:1,000 of Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 and Goat anti-Rabbit IgG (H+L) cross-Adsorbed secondary Antibody, Alexa Fluor 594) for 1 hour at room temperature and then washed. Slides were mounted with Fluorescent mounting medium (10105463, Dako, Glostrup, Denmark) and detected using a Zeiss fluorescence microscope (Image A2 Zeiss, Oberkochen, Germany).