Imaging mass cytometry staining - fresh frozen human brain tissue sections

Note: Use filtered tips for all steps to avoid cross-contamination with metals

Prior to the staining:

1.     Human brain tissue blocks are embedded in Tissue Tek Optimum Cutting Temperature (OCT) medium and 10µm thick sections are cut using a cryostat. 

2.     The frozen tissue sections are mounted on Superfrost Plus glass slides (Knittel Glass) and stored at -80 ̊C until they are stained. 

Day 1

1.     Remove the slide from -80˚C and allow to warm up to room temperature in a closed chamber to avoid condensation

2.     Using a wash bottle, rinse the tissue section with Ultrapure sterile ddH₂O to remove the OCT

3.     Tap slide to discard volume. Dry the slide by dabbing the area around the tissue section with a Kim Wipe 

4.     Use a hydrophobic pen to draw a barrier around the tissue section and allow it to dry. Take care to avoid any leakage of the hydrophobic solution onto the tissue and to avoid breaks along the barrier which would not contain the staining solutions in the subsequent steps

5.     Place the slide inside a humidified chamber and incubate the tissue section in 0.05% PBS-Tween for 20 minutes to re-hydrate 

6.     Tap slide to discard the PBS-Tween. Dry the slide by dabbing the area around the tissue section with a Kim Wipe 

7.     Place the slide inside a humidified chamber and incubate the tissue section in 2-3 drops (sufficient volume to cover the section) of streptavidin blocking solution (Vector, SP-2002). Incubate for 15 minutes at room temperature

8.     Tap slide to discard the streptavidin blocking solution. Transfer the slide to a coplin jar and rinse the tissue section in PBS-Tween (0.05%) for 5 mins at room temperature on a slow-moving shaker

9.     Place the slide inside a humidified chamber and incubate the tissue section in 2-3 drops (sufficient volume to cover the section) of biotin blocking solution (Vector, SP-2002). Incubate for 15 minutes at room temperature

10.  Tap slide to discard the streptavidin blocking solution. Transfer the slide to a coplin jar and rinse the tissue section in PBS-Tween (0.05%) for 5 mins at room temperature on a slow-moving shaker

11.  Place the sections inside a humidified chamber and incubate in 10% normal goat serum solution in PBS for 1 hour at room temperature 

12.  Tap slide to discard the normal goat serum. Dry the slide by dabbing the area around the tissue section with a Kim Wipe 

13.  Place the sections inside a humidified chamber and incubate in SuperBlock (PBS) Blocking Buffer(ThermoFisher, 37518) for 45 minutes at room temperature 

14.  Make up the cocktail of metal-conjugated antibodies (see Table of primary antibodies) in 0.5% BSA in PBS. 

15.  Tap slide to discard the superblock buffer. Dry the slide by dabbing the area around the tissue section with a Kim Wipe 

16.  Place the sections inside a humidified chamber and incubate in the cocktail of metal-conjugated antibodies overnight at 4˚C  

Day 2

17.  Tap slide to discard the antibody cocktail. Transfer the slide to a coplin jar and rinse the tissue section in PBS-0.05% Tween 2X for 8 minutes at room temperature on a slow-moving shaker

18.  Using a wash bottle, further rinse the tissue section with PBS-0.05% Tween

19.  Using a wash bottle, further rinse the tissue section with PBS

20.  Place the slide inside a humidified chamber and stain the tissue section with Ir-intercalator diluted 1/2000 in 0.5% BSA in PBS for 30 minutes at room temperature

21.  Tap slide to discard the Ir-intercalator. Transfer the slide to a coplin jar and quickly rinse the tissue section in Ultrapure sterile ddH₂O for 5 seconds at room temperature on a slow-moving shaker. Do not wash for too long or the signal will dim

22.  Tap slide to discard the water and let the section air dry for 20 minutes

23.  Place the dried stained slide inside a slide box. Place the slide box inside a vacuum-sealed plastic bag with desiccant and store at room temperature until it is imaged.