TITLE: IMC STAINING FOR PARAFFIN SECTIONS 
AUTHORS: Klaus H. Kaestner Lab, Suzanne Shapira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Imaging mass cytometry (IMC) is a multiplexed imaging method that allows samples to be probed with up to forty antibodies at one time. This enables visualization of multiple cell types simultaneously in the native tissue microenvironment. For example, IMC can be applied to study the interactions between endocrine cells and the immune system in pancreata from donors diagnosed with type 1 diabetes. This protocol describes a technique for IMC staining of paraffin sections with a defined antibody panel to identify immune, islet, endocrine, and stromal cell types of the pancreas.</span></div><br/><br/><br/><br/></div>

[STEPS]
?. [Procedure]
Use pressure cooker or heat block to preheat Antigen Retrieval Solution to  before starting.
95 °C
?. [Procedure]
Bake slides in  over for 20 min.
56 °C
?. [Procedure]
Dewax slides in xylene in the fume hood for 5 min X 2.
?. [Procedure]
Hydrate slides in descending grades of ethanol (100%, 95%, 80%, 70%), 5 min each.
?. [Procedure]
While you are dewaxing and hydrating slides, prepare 50 mL 1x PBS by diluting 10x PBS with Milli-Q water.
?. [Procedure]
Rinse slides in PBS for 5 min.
?. [Procedure]
Insert slides with tissues into preheated Retrieval Solution and incubate for 30 min at  .
95 °C
?. [Procedure]
Following incubation, remove from pressure cooker or heat block and place the tube containing the retrieval solution and slides on a lab bench and cool to room temperature, approximately 30 min (or until it reaches room temperature).
?. [Procedure]
Wash the slide with PBS for 10 min.
?. [Procedure]
Use PAP pen to encircle sample.
?. [Procedure]
Block with 3% BSA in PBS for 45 mins at RT.
Note:Use enough blocking solution to cover the section (around 300–500 μL/section).Blocking solution should be diluted from 10% BSA freshly made from powder. Remaining 10% BSA should be aliquoted and stored at   and diluted at the time of use.
?. [Procedure]
To prepare the antibody cocktail, calculate total volume of antibodies (at concentrations specific for the assay) and bring up to final volume with 0.5%BSA in PBS.
Note:Spin Antibody at max speed for 2 minutes and take from the top of the tube to avoid antibody aggregates.Add a small volume of single antibodies into a larger volume of diluent.
?. [Procedure]
Incubate overnight with antibody cocktail at  in hydration chamber (We use a slide box where the slides rest on the shelf and the bottom is covered by wet paper towel).
4 °C
?. [Procedure]
Wash in PBS for 8 min x 2.
?. [Procedure]
Stain the tissue with Ir-Intercalator (1:400) in DPBS for 30 min at RT.
?. [Procedure]
Rinse in ddH²O for 5 min.
?. [Procedure]
Air dry the slide for at least 20 minutes at RT.
Note:A subset of images available on PANC-DB, in the donor specific tabs here are available for viewing asECM, Immune, and Endocrine merged overlays (on the donor-specific tabs above, navigate to Imaging mass cytometry tab and useNext/previous donor buttons to move to other donors).These images go though hot-pixel removal before channel merging as part of the data analysis stepThe remainder full set of files available for download are original unaltered files
?. [List of IMC panel]
1. Panels used currently2. Panel combinations/versions used so far
Anti-Human CD57 (HCD57)-142Nd— 100 TestsAnti-Human CD14 (EPR3653)-144Nd —25 μgAnti-Human Nestin (196908)-146Nd— 100 TestsAnti-Human Pan- Keratin (C11)- 148Nd—25 μgAnti-Human/Mouse CD11b (EPR1344)- 149Sm—25 μgAnti-Human/Mouse CD44 (IM7)-150Nd —100 TestsAnti-Human CD45 (CD45-2B11)- 152Sm—25 μgAnti-Human CD45 (D9M8I)-152Sm— 25 μgAnti-β-actin (2F1-1)- 154Sm—25 μgAnti-Human FoxP3 (236A/E7)-155Gd— 25 μgAnti-Human CD4 (EPR6855)-156Gd —25 μgAnti-Human CD68 (KP1)-159Tb—25 μgAnti-Human CD20 (H1)-161Dy—25 μgAnti-Human CD8a (C8/144B)-162Dy— 25 μgAnti-Human CD8a (D8A8Y)-162Dy— 25 μgAnti-Human pNFkBp65 [S529] (K10x)-166Er—50 Tests