Duolink in situ orange starter kit goat rabbit

Proximity ligation assay (PLA) was performed for in situ detection of the distance between ER and mitochondria (distance < 40 nm) using Duolink^® In Situ Orange Starter Kit Goat/Rabbit (Sigma Aldrich, Germany) following the manufacturer’s instructions. Briefly, two primary antibodies raised in different species (goat anti-calnexin as an ER marker and rabbit anti-TOM20 or rabbit anti-VDAC1 as mitochondrial markers, antibody information in Table 1) were used. A pair of oligonucleotide-labeled secondary antibodies (PLA probes) was added to bind to the primary antibodies. Hybridizing connector oligos were then used to join the PLA probes. Upon close proximity, a circular DNA template is formed by ligase activity resulting in rolling-circle amplification. Amplified signal tethered to the PLA probe was generated that allowed signal detection. The labeled oligos that hybridized to the complementary sequences within the amplicon were visualized and quantified by microscopy image analysis. Leica M205 FA fluorescent stereoscope (Leica Microsystems, USA) and LAS-X-Core Software (3.7.4. version, LAS X Life Science, USA) were used. PLA results were quantified by Image J software (version 1.52a, NIH, USA) following user guide (https://imagej.nih.gov/ij/docs/guide/user-guide.pdf; https://www.unige.ch/medecine/bioimaging/files/1914/1208/6000/Quantification.pdf).

PLA was performed using Duolink In Situ Orange Starter Kit Goat/Rabbit (DUO92106; Sigma-Aldrich), following the manufacturer’s instructions. Images were captured using LSM 700 (Carl Zeiss Micro Imaging) with a 63× objective and analyzed with ImageJ (National Institutes of Health).

PLA was performed using the Duolink In Situ Orange Starter Kit Goat/Rabbit (DUO92106, Sigma-Aldrich) according to the manufacturer's instructions. Briefly, tumor sections of B16F10 melanoma cells as above were fixed in ice-cold acetone for 5 min, washed with PBS, and blocked with 1% BSA in PBS for 2 h at room temperature. 1% BSA in PBS was used for diluting primary antibodies and PLA probes. The tumor sections were incubated with rabbit anti-mouse TM lectin-like domain and goat anti-fibronectin (sc-6952) primary antibodies for 90 min at room temperature. After washing with PBST, the sections were incubated with anti-rabbit MINUS and anti-goat PLUS PLA probes for 60 min at 37°C. The sections were washed in 1 × wash buffer A and then incubated with ligation solution for 30 min at 37°C. After washing with 1 × wash buffer A, the sections were incubated with amplification solution for 100 min at 37°C. The samples were washed in 1 × wash buffer B and in 0.01 × wash buffer B and then mounted with Duolink In Situ Mounting Medium with DAPI. Images were taken using an Olympus confocal microscope with a × 40 objective at a scan speed of 12.5 μs/pixel.