TITLE: Multiplex Immunofluorescence on Fresh Frozen Tissue-V2
AUTHORS: Maya   Brewer, Liz  McDonough, Yuantee  Zhu, Elizabeth  Neumann, Mark  De Caestecker, Danielle  Gutierrez, Jeff  Spraggins

[DESCRIPTION]
Scope:
To describe the procedure for multiple cycles of immunofluorescence on human kidney tissue embedded in carboxymethylcellulose.

Expected Outcome:
Kidney tissue sections that have been tagged with antibodies for imaging microscopy.


[STEPS]
SECTION: Immunofluorescence
1. Place frozen slides from the -80˚C freezer directly into the formalin, and post-fix for 5 minutes.
SECTION: Immunofluorescence
2. Pour used formalin into the appropriate waste container, then fill jar with 1X PBS and wash sections for 5 minutes four times.
SECTION: Immunofluorescence
3. Using a hydrophobic pen, draw a large barrier around each section. Do not allow pen to touch section.
SECTION: Immunofluorescence
5. Incubate sections for 5 minutes with 50 mM glycine (dilute stock in 1X PBS). This reduces autofluorescence by reducing free aldehyde groups.
SECTION: Immunofluorescence
6. Remove glycine, and wash sections in PBS for 5 minutes twice.
SECTION: Immunofluorescence
7. Block sections for 30 minutes with 1X Universal Blocking Reagent (UBR) at room temperature.
SECTION: Immunofluorescence
7.1. Dilute 10X blocking reagent to 1X using 9-parts ddH2O to 1-part UBR.
SECTION: Immunofluorescence
8. Dilute primary antibody to desired working concentration in Antibody Diluent Reagent during blocking step. 
SECTION: Immunofluorescence
9. Add diluted antibody to section and incubate overnight at 4˚C.
SECTION: Immunofluorescence
10. Remove solution, and wash sections with PBS for 5 minutes twice.
SECTION: Immunofluorescence
11. If primary antibody is directly conjugated with a fluorophore, skip to #16.
SECTION: Immunofluorescence
12. If using indirect immunofluorescence, dilute fluorophore-conjugated secondary antibody using Antibody Diluent Reagent.
SECTION: Immunofluorescence
13. Add antibody solution to sections and incubate for 60 minutes at room temperature.
SECTION: Immunofluorescence
14. Remove solution, and wash sections with PBS for 5 minutes three times. 
SECTION: Immunofluorescence
15. Incubate sections in Hoechst 33342 (1:5,000 dilution of 20mM solution in 1X PBS) for 10 minutes.
SECTION: Immunofluorescence
16. Remove Hoechst 33342, and wash sections with PBS for 5 minutes twice.
SECTION: Immunofluorescence
17. Mount slides in 50% glycerol in PBS. Do not seal the coverslips as they will need to be removed later. 
SECTION: Immunofluorescence
17.1. Because slides are not sealed, they must be kept horizontal to prevent the coverslip from falling off, and in the humidified chamber to keep them from drying out.
SECTION: Immunofluorescence
18. Image
SECTION: Immunofluorescence
19. Store slides at 4˚C in a moisture chamber.
SECTION: De-Coverslipping
20. Remove coverslip from sections by incubating slides in a vertical staining jar filled with PBS for 30 minutes with slight agitation (i.e. plate rocker).
SECTION: De-Coverslipping
21. Slowly lift slide from vertical jar and allow coverslip to release from slide via gravity.
SECTION: De-Coverslipping
22. Wash slide in PBS for 5 minutes three times to remove any residual glycerol.
SECTION: De-Coverslipping
22.1. Place slides back in vertical staining jar full of PBS with slight agitation to wash.
SECTION: Fluorophore Inactivation
23. 
Prepare basic solution of 0.5 M sodium bicarbonate (NaHCO3), pH 11.2 (acceptable range: 10.9-11.3).
SECTION: Fluorophore Inactivation
25. Add inactivating solution to each section and incubate at room temperature for 15 minutes in the presence of white light (LED light).
  

SECTION: Fluorophore Inactivation
25.1. For this, place slides on a plastic surface on top of the LED light. We often use multiple plates for this.
SECTION: Fluorophore Inactivation
25.2. Repeat this process with fresh solution four more times.
SECTION: Fluorophore Inactivation
26. Remove solution and wash sections in PBS for 5 minutes four times.
SECTION: Fluorophore Inactivation
26.1. After inactivating the fluorophores, slides are mounted in 50% glycerol and imaged to confirm complete fluorophore inactivation, followed by the removal of the coverslip, and three 5 minutes washes in PBS. Hoechst stain will not bleach and is necessary for image registration later.
SECTION: Subsequent Immunofluorescence Cycles
27. Dilute fluorophore-conjugated primary antibodies using Antibody Diluent Reagent.
SECTION: Subsequent Immunofluorescence Cycles
28. Add diluted antibodies to each section and incubate overnight at 4˚C.
SECTION: Subsequent Immunofluorescence Cycles
29. Remove antibody solution and wash sections with PBS for 5 minutes three times.
SECTION: Subsequent Immunofluorescence Cycles
30. Incubate sections in Hoechst (1:5,000 dilution in 1X PBS) for 5 minutes (this may not be necessary, but does no harm).
SECTION: Subsequent Immunofluorescence Cycles
31. Remove Hoechst, and wash sections with PBS for 5 minutes twice.
SECTION: Subsequent Immunofluorescence Cycles
32. Mount slides in 50% glycerol in PBS.
SECTION: Subsequent Immunofluorescence Cycles
33. Image
SECTION: Subsequent Immunofluorescence Cycles
34. Store slides at 4˚C in a moisture chamber.
35. Sections II-IV are repeated for each remaining cycle.
SECTION: Immunofluorescence
4. Place slides in humidified chamber and cover section in PBS by using a pipettor so that they do not dry.
SECTION: Immunofluorescence
4.1. To remove solutions from sections that are in the humidified chamber, tip the solution off of the slide into the chamber.  

SECTION: De-Coverslipping
21.1. If the coverslip does not release from the slide, return the slide to PBS for an additional 15 minutes.
SECTION: Fluorophore Inactivation
23.1. Mix 21 g of sodium bicarbonate with 500 ml ddH2O thoroughly.
SECTION: Fluorophore Inactivation
23.2. Using sodium hydroxide pellets, bring the solution to a pH 11.2 (acceptable range: 10.9-11.3). Measure the pH each week it is used and adjust as needed.
SECTION: Fluorophore Inactivation
23.3. Solution can be stored at 4C for 6 months.
SECTION: Fluorophore Inactivation
24. Prepare a solution of 0.1 M NaHCO3, 3% H2O2 in ddH2O.
SECTION: Fluorophore Inactivation
24.1. As an example, to make up 10 ml of inactivation solution, add 2 ml of 0.5 M NaHCO3and 7 ml ddH2O.
SECTION: Fluorophore Inactivation
24.2. Solution is aliquoted into five tubes (1.8 ml per tube).
SECTION: Fluorophore Inactivation
24.3. 200 µl of hydrogen peroxide is added to each tube prior to the solution being added to the section. Solution must be made fresh and added to sections immediately after the addition of H2O2. This reaction is time sensitive.
SECTION: Quality Controls
36. Controls: When testing new antibodies, extra sections are used for controls to determine the autofluorescence and specificity of each antibody.
SECTION: Quality Controls
36.1. Negative control: Used to observe autofluorescence of the tissue. No primary or secondary antibody is added to the section.
SECTION: Quality Controls
36.2. Isotype control: Checks for any non-specific interactions of the primary antibody. A non-immune antibody of the same species and isotype (i.e. mouse IgG1) of the primary antibody being tested is incubated at the same concentration. A secondary antibody is added.
SECTION: Quality Controls
36.3. Positive control: Most antibodies used have targets that are found in kidney tissue in high amounts. For the immune cell markers used within this project, the antibodies are first tested in spleen tissue to confirm they work. They are then validated in kidney tissue.
SECTION: Antibody Validation
37. All antibody validation is done on the same tissue block. When that block is no longer available, all antibodies must be validated on the new block.
SECTION: Antibody Validation
38. Working dilutions are determined by the recommendations from the company. A series of dilutions are tested based on these recommendations. 
SECTION: Antibody Validation
38.1. If there are no references from the company, antibody dilutions range from 2-20 µg/mL depending on the concentration of the stock.
SECTION: Antibody Validation
39. Directly conjugated antibodies are validated in the same steps as those that are unconjugated. A secondary antibody in a different fluorophore from the direct conjugate is added to confirm the conjugation.
SECTION: Antibody Validation
39.1. The working dilution for directly conjugated antibodies is determined based on the specificity, intensity (secondary vs. direct conjugate), and signal: noise ratio.
SECTION: Antibody Validation
39.2. Directly conjugated antibodies are tested for the ability to withstand one freeze/thaw cycle. This is completed by freezing a small aliquot of the conjugated antibody at -20C. This aliquot is thawed and stained on a section adjacent to a section stained with an aliquot of the antibody that has not been frozen.
SECTION: Antibody Validation
39.3. All antibodies conjugated in the lab are validated via quantifying the moles of dye: moles of protein (acceptable range: 0.7-2). See Antibody Purification and Labeling Protocol for details.
SECTION: Antibody Validation
40. Each antibody is evaluated and validated using other antibodies to determine the sensitivity and selectivity. They are then placed in specific groups based on the imaging results.
SECTION: Antibody Validation
40.1. Enhanced (orthogonal): Co-labeling with a different antibody to the same antigen.
SECTION: Antibody Validation
40.2. Approved: Different antigens, yet the cell expression is the same.
SECTION: Antibody Validation
40.3. Supported: Localization is the right cell type, but there is not a second antibody for validation.
SECTION: Antibody Validation
41. New lots of antibodies are validated by staining the old lot while simultaneously staining the new lot using a series of dilutions including the optimized dilution of the old lot.
SECTION: Multiplexing Qc
42. Prior to multiplexing, each cycle is stained independently and imaged.
SECTION: Multiplexing Qc
43. The antibodies are compared to when they were previously stained alone to ensure the specificity and intensity remains the same. 
SECTION: Multiplexing Qc
44. A practice run of MxIF must be successfully completed prior to starting an experiment.