Jul 26, 2024
Immunohistochemistry - uPAR in mouse lung tissue
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Sojin Kim 2024. Immunohistochemistry - uPAR in mouse lung tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v923dql3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: July 11, 2024
Last Modified: July 26, 2024
Protocol Integer ID: 103246
Abstract
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.
Materials
Tissue preparation
- Xylene
- Ethanol
- PBS (pH 7.5)
Antigen Retrieval
- Graduated cylinder (for measuring 1.6 L)
- Serologic pipette (for measuring 15 mL)
- Timer
- PBS (pH 7.5)
For the pressure cooker method:
- Pressure cooker
- Metal staining rack
For the microwave method:
- Microwave
- Plastic Coplin jar and staining rack
Quench/Block
- BLOXALL Endogenous Blocking Solution, Peroxidase and Alkaline Phosphatase (Vector Laboratories, Cat.# SP-6000-100, link)
Primary antibody
- Bovine Serum Albumin (BSA), Immunohistochemical Grade (Vector Laboratories, Cat.# SP-5050-500, link)
Secondary antibody
Substrate/Chromogen
Counterstain
Coverslip/Mount
- Cover Slip, Square, #1.5 Corning 0211 Glass, 22 x 22mm (Thomas Scientific, Cat.# 1139W03, link)
Tissue Preparation
Tissue Preparation
Cut and mount sections on slides (The thickness of sectioning: 5 µm ))
Deparaffinize and Rehydrate
Deparaffinize and Rehydrate
Place the slide in a 56 °C to 60 °C oven for 00:15:00
15m
Incubate as below:
- Xylene, 00:05:00
- Xylene, 00:05:00 . Shake off excess liquid
- 100% EtOH, 00:03:00
- 100% EtOH, 00:03:00 . Shake off excess liquid
- 90% EtOH, 00:03:00 . Shake off excess liquid
- 80% EtOH, 00:03:00
- Rinse the slides in gently running tap water for00:00:30 (avoid a direct jet which may wash off or loosen the section)
- Place in PBS wash bath for further rehydration (00:30:00 , Room temperature )
52m 30s
Mark the tissue area with an ImmPrint Histology Pen.
Antigen Retrieval
Antigen Retrieval
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.
Option 1. Pressure Cooker
Position slides into metal staining racks. Do not place slides close together; uneven staining may occur.
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)
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.
Start timing once the cooker has pressurized start timing. After 00:01:00 , 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.
1m
Open the lid, remove slides, and place immediately into a tap water bath.
DO NOT LET SECTIONS DRY OUT.
Wash sections in PBS buffer (7.5 ) for 00:05:00
5m
Option 2. Microwave
Position slides in a plastic rack (do not use standard glass histology rack and vessel)
Add the appropriate antigen retrieval buffer to the rack. Do not tighten the lid.
Set it to full power and wait until the solution comes to a boil. Boil for 00:20:00 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.
20m
Remove the vessel and run cold tap water into it for 10 min. Use care with a hot solution.
Quench/Block
Quench/Block
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.
Apply Blocking solution (Vector Laboratories, Cat.# SP-6000) to tissue sections and incubate for 00:10:00 Room temperature
Appropriate care should be exercised when using this reagent to avoid contact with eyes and skin. Dispose by local regulations
10m
Wash sections with PBS for 00:05:00
5m
Primary Antibody
Primary Antibody
How to choose a primary antibody
- Specific for antigen of interest
- Consider tissue species and preparation (fixation)
- Consider antigen retrieval requirements
Allow the slides to drain, shake off excess fluid with a brisk motion, and carefully wipe each slide around the sections.
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)
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.
Incubate for at least 01:00:00 . at 37 °C in humidified chamber. Longer incubations are advised for low-density antigens.
1h
Rinse gently with PBS from a wash bottle. Place the slide in a PBS wash bath for 00:05:00 .
5m
Secondary Antibody
Secondary Antibody
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
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.
Incubate for 00:30:00 at Room temperature in a humidity chamber
30m
Rinse gently with PBS from a wash bottle. Place the slide in a PBS wash bath for 00:05:00
5m
Substrate/Chromogen
Substrate/Chromogen
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.
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.
Allow each slides to drain. Shake off excess fluid and carefully wipe the slide as before.
Incubate with the substrate working solution at Room temperature for 00:02:00 to 00:10:00 . Optimal development times should be determined by the investigator.
12m
Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle.
Wash for 00:05:00 in water.
5m
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.
Counterstain
Counterstain
Rinse the slide in tap water.
Incubate sections with Hematoxylin QS for 00:00:05 to 00:00:45
50s
Rinse sections with running tap water until rinse water is colorless or dip for approximately 00:00:10 in water.
10s
Coverslip/Mount
Coverslip/Mount
1m
1m
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).
Perform rapid dehydration with two washes in 99% histological grade isopropanol (also called 2-propanol or isopropyl alcohol) for at least 00:01:00 each. 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.
1m
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 ).
Slowly lower coverslip onto mounting medium avoiding air bubbles.
Allow slides to dry flat at ambient temperature.
Slides can be viewed immediately. However, 00:10:00 to 00:20:00 are required for coverslip to set.
30m
Store coverslipped slides at ambient temperature. We do not recommend storage at elevated temperature (oven).
Protocol references
Vector Laboratories - Immunohistochemistry Resource Guide
Millipore Sigma - Immunohistochemistry Procedure for Paraffin-Embedded Tissues