Liver tissue staining with multiple lanthanides-tagged antibodies

Cut the fresh frozen sections at 5µm using a cyromicrotome and mount them on Au-coated silicon slides

Place the slides at -20 ◦C for 1h and then at 4 ◦C for 30 min for temperature equilibrium

Fix the tissue slides in 1% PFA for 5 min at 4 ◦C, then in prechilled methanol for 5 min at -20 ◦C

Wash the slides twice for 5 min in a glass petri dish by submerging the slides in 25 ml wash buffer, following by rehydrating the slides for 5 min in a glass container with 25ml DPBS and rinsing the slides for 5 min in a glass container with 25ml wash buffer

Use the Liquid-Repellent Slide Marker Pen to draw a circle around the tissue section to create a barrier to contain the solutions on the tissue sections

Apply 100 µl 3% goat serum solution to each slide for 1 h at RT and then remove excess block solution by tapping on a tissue

Prepare the antibody cocktail. Vortex all the antibodies for 15 s and then centrifuge at 12,000G for 2 min, followed by diluting the upper liquid of each antibody in 0.5% PBS as desired concentration

Add 100 µl of the antibody cocktail to each section and incubate overnight at 4◦C in a sealed glass petri dish in a fridge

After the incubation, wash the sections two times for 5min in a glass petri dish with 5 ml wash buffer with slow agitation

Incubate the slides with 100 µl 1:100 dilution of Intercalator-Ir in DPBS for 30 min at RT

After nuclei staining, wash the slides for 5 min in a glass container with 5 ml DPBS

Quickly dip the slides in a container with 5 ml DI water for 15 s, repeat three times

Dry the slides in a fume hood at RT for 20 min

Store the slides at 4◦C until C60-SIMS imaging.