Alexa fluor 594 conjugated goat anti rat secondary antibody

After the HCR step and rinsing with 5× SSCT for 5 min, slides were incubated in blocking buffer (5% bovine serum albumin in TBS) for 30 min at room temperature in a humid chamber. A rat anti-α-tubulin (Bio-Rad) primary antibody was diluted 1:500 in blocking buffer and incubated for 1 h at room temperature in darkness. Slides were washed three times in TBS. Alexa Fluor 594-conjugated goat anti-rat secondary antibody (Thermo Scientific) was diluted 1:1,000 in blocking buffer containing DAPI (200 ng/ml), and slides were incubated for 1 h at room temperature in darkness. Samples were washed three times in TBS, dried, mounted in ProLong Diamond antifade mountant (Thermo Scientific), and stored at 4°C in darkness.

Immunofluorescence staining of cryosections was performed on 12 µm thick cryosections. Tissue sections were fixed by immersing the slides in pre-cooled acetone (−20 °C) for 10 min. Slides were removed from fixative and acetone was allowed to evaporate from the tissue sections for 20 min. After slides were rinsed in PBS, cryosections were blocked in blocking buffer (5% serum in PBS) for 1 h at room temperature. Anti-CD8 primary antibody diluted in blocking buffer (1:100) was added to the slides and kept in a humidified chamber for 2 h at room temperature. Slides were rinsed twice in PBS before adding Alexa Fluor 594 conjugated goat anti-rat secondary antibody (ThermoFisher Scientific, A48264) diluted 1:500 in blocking buffer and incubated for 1 h at room temperature. Slides were then rinsed three times in PBS before mounting with Vectashield DAPI hard set (Vector, H-1500). Cryosections were analysed using a fluorescence microscope (Olympus BX51) and images were obtained at 20× magnification.