TITLE: Immunohistochemistry - uPAR in mouse lung tissue
AUTHORS: Sojin Kim

[DESCRIPTION]
Immunohistochemistry (IHC) is a method to detect specific target antigens (proteins) in tissue sections using antibodies. Immunocytochemistry (ICC) uses similar techniques to localize cellular proteins in cell preparations. Both IHC and ICC are powerful tools that provide insights into gene expression, spatial
relationships, and biomarker identification in various applications. These applications include basic research, assessment of normal and disease states within human and animal tissues, and assessment of plant pathology. The target antigen, bound by the detection antibody, is visualized using chromogenic or fluorescence detection. In chromogenic detection, the detection antibody is conjugated to an enzyme. The enzyme, usually horseradish peroxidase or alkaline phosphatase, catalyzes the conversion of its respective chromogen to a colored precipitate at the site of the antigen. This precipitate can be visualized by using brightfield microscopy. Certain chromogens can also be visualized by using electron, darkfield or fluorescence microscopy. In fluorescence detection, the detection antibody is conjugated to a fluorophore, which can be visualized using fluorescence microscopy. 

[STEPS]
SECTION: Tissue Preparation
1. Cut and mount sections on slides (The thickness of sectioning:  )) 

SECTION: Antigen Retrieval
5. Tris-based vs. Citrated-based antigen retrieval reagents
Antibody/Epitope Sensitivity: Depending on the sensitivity of the specific antigen or antibody being targeted, one might be preferred over the other. Some epitopes might only be retrievable in acidic or alkaline conditions.
Tissue Type: Different tissues may respond better to different pH conditions for optimal antigen retrieval.
SECTION: Quench/Block
11. How to choose a blocking reagents
Blocking agents minimize background signals from endogenous enzyme activity, biotin, and non-specific binding of tissue elements (charged particles, macromolecules, Fc receptors) with detection reagents.
SECTION: Primary Antibody
14. How to choose a primary antibody
Specific for antigen of interest
Consider tissue species and preparation (fixation)
Consider antigen retrieval requirements
SECTION: Secondary Antibody
20. How to choose a secondary/Tertiary antibody
Choose HRP or AP enzyme system
Consider sensitivity requirements
Consider species of primary antibody
Consider tissue species

Consider species cross-reactivity
When choosing the optimal detection system for your application, it is important to consider not only the species of the primary antibody but also the species of the tissue under examination. If the species of the primary antibody and the species of the tissue are closely related (for example, rat and mouse), the biotinylated secondary antibody may cross-react with endogenous IgG in the tissue section. This can lead to background staining. The following options may minimize background staining in these instances:
Directly label the primary antibody with biotin
Use a biotinylated secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely-related species
Use the M.O.M. (Mouse on Mouse) Immunodetection System for applications of mouse primary antibodies on mouse tissue

4 Options
 

Option A: Convenient. Consistent. Ready-to-use. Non-biotin based
Option B: Dual label (two antigen detection). Convenient. Consistent. Ready-to-use. Non-biotin based
Option C: Economical. Biotin-based
Option D: Highest sensitivity. Non-biotin-based
SECTION: Substrate/Chromogen
24. How to choose enzyme substrate
Color
Compatibility with other system reagents (counterstains, mounting media, and other substrates for multiplexing)

 

How to choose nuclear counterstain
A counterstain introduces color to specific cellular structures to provide contrast to the colored enzyme substrate. Counterstaining aids in visualization and target localization, facilitating interpretation of morphology and cell structure within the tissue section. Our nuclear counterstains are packaged as convenient, ready-to-use solutions for use on individual slides or in staining dishes. 
Blue, green, or red compatibility with substrate, mounting media 

 

HRP vs AP
HRP (peroxidase-based) detection system is a preferred choice for many IHC applications that
require sharp, distinct localization of the target antigen. 
AP (Alkaline phosphatase-based) detection kit is a good alternative to using peroxidase-based
reagents in specimens that exhibit problematic levels of endogenous peroxidase activity. 
SECTION: Coverslip/Mount
35. How to choose mounting media
Aqueous vs non-aqueous
Compatibility with substrate(s) and counterstain
 
For permanent, non-aqueous mounting: Dehydrate, clear and coverslip using a non-aqueous mounting media, such as VectaMount‱ Mounting Medium (H-5000) or VectaMount‱ Express Mounting Medium
(H-5700).
For aqueous mounting: Coverslip using an aqueous mounting media such as VectaMount‱ AQ Mounting Medium (H-5501).

SECTION: Antigen Retrieval
6.1. Position slides into metal staining racks. Do not place slides close together; uneven staining may occur.
SECTION: Antigen Retrieval
6.2. Pour 1600 mL of distilled water into a stainless steel pressure cooker. 
Shake well before use, and then add 15 mL of the concentrated stock Antigen Unmasking Solution (Vector Laboratories, Cat.# H-3300-250)
SECTION: Antigen Retrieval
6.3. Cover, but do not lock the lid of a pressure cooker, and bring a solution to a boil.
Place slides in a pressure cooker, ensuring slides are well immersed in diluted Antigen Unmasking Solution. Lock lid. 
SECTION: Antigen Retrieval
6.4. Start timing once the cooker has pressurized start timing. After 1 min, remove the pressure cooker from a heat source and run it under cold water. 
DO NOT OPEN LID UNTIL THE INTERNAL PRESSURE HAS BEEN COMPLETELY REDUCED.
SECTION: Antigen Retrieval
6.5. Open the lid, remove slides, and place immediately into a tap water bath.
DO NOT LET SECTIONS DRY OUT.
SECTION: Antigen Retrieval
6.6. Wash sections in PBS buffer (pH 7.5) for 5 min
SECTION: Deparaffinize and Rehydrate
4. Mark the tissue area with an ImmPrint Histology Pen.
SECTION: Deparaffinize and Rehydrate
2. Place the slide in a 56 °C to 60 °C oven for 15 min 

SECTION: Quench/Block
12. Apply Blocking solution (Vector Laboratories, Cat.# SP-6000) to tissue sections and incubate for 10 min Room temperature 
Appropriate care should be exercised when using this reagent to avoid contact with eyes and skin. Dispose by local regulations
SECTION: Quench/Block
13. Wash sections with PBS for 5 min 

SECTION: Secondary Antibody
21. Apply a secondary antibody matched to the primary antibody.
For uPAR: Apply Horse Anti-Goat IgG Antibody (H+L), Biotinylated (Vector Laboratories, Cat.# BP-9500-50) to the tissue section.
SECTION: Secondary Antibody
22. Incubate for 30 min at Room temperature in a humidity chamber
SECTION: Substrate/Chromogen
25. It is recommended to prepare the substrate mixture during the final wash step.
Please check the table above and choose the appropriate one.

For uPAR: Add 1 drop of ImmPACT DAB Reagent 1 to 1 mL ImmPACT DAB Diluent. Mix well before use.

IMPORTANT: DAB is a suspected carcinogen. Appropriate care should be exercised when using this reagent including gloves, eye protection, lab coats, and good laboratory procedures. Dispose in accordance with local regulations.
SECTION: Substrate/Chromogen
27. Incubate with the substrate working solution at Room temperature for 2 minto 10 min . Optimal development times should be determined by the investigator.
SECTION: Substrate/Chromogen
29. Wash for 5 min in water.
SECTION: Substrate/Chromogen
30. Notes
An unused working solution is stable for up to 14 days if stored at 2–8 °C or up to 5 days if stored at room temperature (approximately 23 °C). The working solution may change color during storage, but this will have no effect on the quality or intensity of the staining. No sample-obscuring precipitate will form.
It is not necessary or recommended to add detergent to ImmPACT DAB working solution to reduce surface tension (e.g., for use in an automated stainer).
The DAB reaction product can be intensified using a DAB Enhancing Solution (H-2200) after development.
SECTION: Counterstain
31. Choose counterstain based on this information (link)
SECTION: Deparaffinize and Rehydrate
3. Incubate as below: 
Xylene, 5 min 
Xylene, 5 min . Shake off excess liquid
100% EtOH, 3 min 
100% EtOH, 3 min . Shake off excess liquid
90% EtOH, 3 min . Shake off excess liquid
80% EtOH, 3 min
Rinse the slides in gently running tap water for30 s (avoid a direct jet which may wash off or loosen the section)
Place in PBS wash bath for further rehydration (30 min, Room temperature)

SECTION: Primary Antibody
15. Allow the slides to drain, shake off excess fluid with a brisk motion, and carefully wipe each slide around the sections.
SECTION: Primary Antibody
16. Dilute the primary antibody or negative control reagent to its optimal dilution in diluent (100 uL/tissue sections). The diluent alone may be used as a negative control. 
For uPAR: Prepare 1:50 and 1:100 antibody dilution in BSA (Vector Laboratories, Cat.# SP-5050-500)
SECTION: Primary Antibody
17. Apply 100 µL primary antibody solution to the appropriate slides, covering the tissue sections.
Tilt each slide in two different directions so the liquid is spread evenly over the slide.
SECTION: Primary Antibody
18. Incubate for at least 60 min. at 37 °C in humidified chamber. Longer incubations are advised for low-density antigens.
SECTION: Primary Antibody
19. Rinse gently with PBS from a wash bottle. Place the slide in a PBS wash bath for 5 min.
SECTION: Secondary Antibody
23. Rinse gently with PBS from a wash bottle. Place the slide in a PBS wash bath for 5 min
SECTION: Substrate/Chromogen
28. Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle.
SECTION: Substrate/Chromogen
26. Allow each slides to drain. Shake off excess fluid and carefully wipe the slide as before.
SECTION: Antigen Retrieval
6. Option 1. Pressure Cooker 
SECTION: Antigen Retrieval
7. Option 2. Microwave 
SECTION: Antigen Retrieval
7.1. Position slides in a plastic rack (do not use standard glass histology rack and vessel)
SECTION: Antigen Retrieval
8. Add the appropriate antigen retrieval buffer to the rack. Do not tighten the lid.
SECTION: Antigen Retrieval
9. Set it to full power and wait until the solution comes to a boil. Boil for 20 min from this point. Use a non-sealed vessel to allow for evaporation during the boil. Be sure to monitor for evaporation and watch out for boiling over during the procedure and do not allow the slides to dry out.

SECTION: Antigen Retrieval
10. Remove the vessel and run cold tap water into it for 10 min. Use care with a hot solution.
SECTION: Counterstain
32. Rinse the slide in tap water.
SECTION: Counterstain
33. Incubate sections with Hematoxylin QS for 5 s to 45 s 
SECTION: Counterstain
34. Rinse sections with running tap water until rinse water is colorless or dip for approximately 10 s in water.
SECTION: Coverslip/Mount
36. Perform rapid dehydration with two washes in 99% histological grade isopropanol (also called 2-propanol or isopropyl alcohol) for at least 1 mineach. Histological-grade alcohol can also be used. However, isopropanol is recommended. Processing through a standard graded series of alcohol can also be substituted if desired.

SECTION: Coverslip/Mount
37. Remove a slide from the isopropanol, blot excess, and immediately apply mounting medium to the tissue section(s)*. Two drops of mounting medium are recommended for a standard-sized tissue section (less than 22 mm2 ).
SECTION: Coverslip/Mount
38. Slowly lower coverslip onto mounting medium avoiding air bubbles.
SECTION: Coverslip/Mount
39. Allow slides to dry flat at ambient temperature.
SECTION: Coverslip/Mount
40. Slides can be viewed immediately. However, 10 min to  20 min are required for coverslip to set. 

SECTION: Coverslip/Mount
41. Store coverslipped slides at ambient temperature. We do not recommend storage at elevated temperature (oven).