Aug 11, 2023
In situ immunoglobulin G (IgG) detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues
- Jayne E Wiarda1,
- Crystal Loving1
- 1National Animal Disease Center, ARS, USDA
Protocol Citation: Jayne E Wiarda, Crystal Loving 2023. In situ immunoglobulin G (IgG) detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdjxzlmk/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: Working
We use this protocol and it's working
Created: August 11, 2023
Last Modified: August 11, 2023
Protocol Integer ID: 86386
Funders Acknowledgement:
USDA-ARS
Grant ID: CRIS 5030-31320-004-00D
Abstract
An immunohistochemistry (IHC) staining protocol for in situ identification of IgG in pig tissue.
Attachments
Guidelines
Assay Controls:
Here are a few controls you can use to ensure assay is working correctly:
- IHC controls:
o Negative
control (primary antibody only)
§ This slide receives 0.05% PBS-T in place of secondary antibody
o Negative control (secondary antibody only)
§ This slide receives 1% BSA in PBS in place of diluted primary antibody
o Batch control
§ If performing staining across multiple batches, include serial sections of one tissue in each batch that has positive staining for Salmonella
Assay variations:
Parameters for some steps (e.g. antibody incubations, antigen retrieval, chromogen incubations, counterstaining) may need to be further optimized for different tissues or targets.
Materials
Equipment:
- Pipettes/pipette tips – volumes ranging between 2-1000 uL
- Drying oven (able to reach & hold 60℃)
- Fume hood
- Slide staining tray (e.g. Simport M920-2)
- Tissue-Tek Vertical 24 slide rack (American Master Tech Scientific LWS2124)
- Tissue-Tek Staining Dishes (American Master Tech Scientific LWS20WH)
- Tissue-Tek Clearing Agent Dishes, xylene resistant (American Master Tech Scientific LWS20GR)
- Bright field microscope
Reagents/Supplies:
***For all reagents, refer
to MSDS to determine appropriate precautions, personal protective equipment
(PPE), and disposal methods before use***
- Distilled water (obtained in-house)
- 0.05% PBS-Tween (PBS-T), pH 7.35 (made in-house)
- 1% bovine serum albumin (BSA) in PBS (made in-house)
- Xylenes (Macron Fine Chemicals 8668-16)
- 100% ethanol (Pharmco 111000200)
o Dilute with distilled water to make 95%, 85%, and 70% concentrations
- Pro-Par Clearant (Anatech 510)
- Fixative
o 10% NBF (Cancer Diagnostics, Inc. 111)
- ImmEdge Hydrophobic Barrier Pen (Vector H-4000)
- Proteinase K, ready to use (Dako S3020)
- Dual Endogenous Enzyme Block (Dako S2003)
- Protein Block (Dako X0909)
- Rabbit anti-porcine IgG (H+L) Secondary Antibody (Novus Biologicals NBP1-73812)
o Stock concentration unspecified; reconstituted lyophilized antibody in 2 mL distilled water
- EnVision+ System HRP Labelled Polymer Anti-Rabbit (Dako K4003)
- Liquid DAB+ (Dako K346811-2)
o DAB+ Substrate Buffer
o DAB+Chromogen
- Gill’s Hematoxylin I (American Master Tech Scientific HXGHE1LT)
- Refrax Mounting Medium (Anatech 711)
- #1 thickness cover glass (Fisherbrand 12-545-F)
Safety warnings
***For all reagents, refer to MSDS to determine appropriate precautions, personal protective equipment (PPE), and disposal methods before use***
Before start
Starting specimens:
Starting samples = FFPE tissues cut to 4 micron thickness and adhered to positively-charged microscopy slides (e.g. SuperFrost Plus Slides; Fisher Scientific 12-550-15). It is crucial that tissues are adequately fixed to prevent tissue degradation but not over-fixed as to over-fragment RNA. Tissues no thicker than 0.5 centimeters should be freshly harvested and placed into 10% neutral-buffered formalin (NBF) at a ratio of at least 20 volumes fixative per one volume tissue. Tissues should be fixed for between 16-30 hours at room temperature (RT), followed by immediate transfer to 70% ethanol and processing into FFPE tissue blocks. Fixation times should be optimized for individual tissues and experiments.
Baking
Baking
Before starting the assay:
- Preheat a dry oven to 60℃
- Load slides for assay into vertical slide rack
Baking
- Bake slides 20 min 60℃
While slides bake:
- Prepare 0.05% PBS-T (can store at RT up to 1 month)
Deparaffinizing & Rehydrating
Deparaffinizing & Rehydrating
Immediately before deparaffinizing:
Add ~200 mL xylenes to each of three clearing agent dishes in a fume hood
Add ~200 mL 100% ethanol to each of two staining dishes in a fume hood
Add ~200 mL 95% ethanol to a staining dish in a fume hood
Add ~200 mL 85% ethanol to a staining dish in a fume hood
Add ~200 mL 70% ethanol to a staining dish in a fume hood
Add ~200 mL distilled water to a staining dish in a fume hood
Add ~200 mL PBS-T to a staining dish in a fume hood
Deparaffinizing & Rehydrating
- Submerge slide in fresh xylenes 5 min RT
- Submerge slide in fresh xylenes 5 min RT
- Submerge slide in fresh xylenes 5 min RT
- Submerge slides in fresh 100% ethanol 1 min RT
- Submerge slides in fresh 100% ethanol 1 min RT
- Submerge slides in fresh 95% ethanol 1 min RT
- Submerge slides in fresh 85% ethanol 1 min RT
- Submerge slides in fresh 70% ethanol 1 min RT
- Submerge slides in fresh distilled water 3 min RT
- Submerge slides in fresh PBS-T for transport
While slides deparaffinize/rehydrate:
- Turn off dry oven
- Prepare humidified slide staining tray by adding water to bottom & placing lid on top
- Add ~200 mL PBS-T to each of two staining dishes
Hydrophobic Barrier
Hydrophobic Barrier
Hydrophobic Barrier
- Apply hydrophobic barrier around each tissue
o ----------One by one, unload slides from vertical rack submerged in PBS-T. Dry off only the area around the tissue where a barrier will be drawn with a hydrophobic barrier pen. Keep tissue area wet the whole time. Draw barrier and place slide flat in the slide staining tray. Using a pipette, apply a small amount of PBS-T within the barrier (just enough to keep the tissue wet while drawing barriers on remaining slides)
- Leave slides in slide staining tray
Protease Digestion
Protease Digestion
Protease Digestion
- Decant slides and again place flat in slide staining tray
- Incubate with Proteinase K 3 min RT
o ----------Invert bottle immediately before use; apply drops to completely cover tissues; let incubate in slide staining tray with lid closed
- Decant slides and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
Tissue Quenching
Tissue Quenching
Tissue Quenching
- Decant slides and again place flat in slide staining tray
- Incubate with Dual Endogenous Enzyme Block 10 min RT
o ----------Invert bottle immediately before use; apply drops to completely cover tissues; let incubate in slide staining tray with lid closed
- Decant slides and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
While slides incubate with enzyme block:
- Discard deparaffinizing & rehydrating and protease digestion reagents
- Add ~200 mL PBS-T to each of two staining dishes
Protein Blocking
Protein Blocking
Protein Blocking
- Decant slides and again place flat in slide staining tray
- Incubate with Protein Block 20 min RT
o ----------Invert bottle immediately before use; apply drops to completely cover tissues; let incubate in slide staining tray with lid closed
- Decant slides and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
While slides incubate with protein block:
- Discard tissue quenching reagents
- Prepare primary antibody by adding IgG antibody to 1% BSA in PBS at a dilution of 0.02 uL/mL (1:50,000 dilution). Total volume to use is dependent on tissue sizes. Make sure to mix reagents before pipetting.
Primary Antibody
Primary Antibody
Primary Antibody
- Decant slides and again place flat in slide staining tray
- Incubate with diluted primary antibody overnight at 4℃
o ----------Apply drops to completely cover tissues; let
incubate in slide staining tray with lid closed
- Remove slides from slide staining tray, decant, and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
While slides are incubating with primary antibody:
- Discard protein blocking reagents
The next day:
- Add ~200 mL PBS-T to each of two staining dishes
Secondary Antibody
Secondary Antibody
Secondary Antibody
- Decant slides and again place flat in slide staining tray
- Incubate with anti-rabbit HRP polymer 30 min RT
o ----------Invert bottle immediately before use; apply drops to completely cover tissues; let incubate in slide staining tray with lid closed
- Decant slides and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
While slides are incubating with secondary antibody:
- Discard remaining primary antibody reagents
- Add ~200 mL PBS-T to each of two staining dishes
Chromogen Detection
Chromogen Detection
Immediately before chromogen detection:
- Prepare diluted DAB chromogen by adding 1 drop DAB substrate per 1 mL substrate buffer. Total volume to use is dependent on tissue sizes. Make sure to mix reagents thoroughly. Store in the dark due to light sensitivity
Chromogen Detection
- Decant slides and again place flat in slide staining tray
- Incubate with diluted DAB chromogen 2 min RT
o ----------Pipette well to mix immediately before use; pipette appropriate volumes to completely cover tissues & let incubate in slide staining tray with lid closed
- Decant slides and transfer to vertical slide rack
- Submerge slide rack in fresh PBS-T 2 min RT
- Submerge slide rack in fresh PBS-T 2 min RT
While slides are incubating with DAB chromogen:
- Discard secondary antibody reagents
- Add ~200 mL PBS-T to each of two staining dishes
- Add ~200 mL 25% hematoxylin to one staining dish
o ----------Prepare by combining 150 mL distilled water with 50 mL Gill’s Hematoxylin
- Add ~200 mL distilled water to each of three staining dishes
- Add ~200 mL 95% ethanol to a staining dish in a fume hood
- Add ~200 mL 100% ethanol to each of three staining dishes in a fume hood
- Add ~200 mL Pro-Par to each of three clearing agent dishes in a fume hood
Counterstaining
Counterstaining
Counterstaining
- Submerge slide rack in diluted hematoxylin 15 sec RT
- Submerge slide rack in fresh distilled water, dunking 3-5 times
- Submerge slide rack in fresh distilled water, dunking 3-5 times
- Submerge slide rack in fresh distilled water, dunking 3-5 times
Mounting
Mounting
Mounting
- Submerge slides in fresh 95% ethanol 1 min RT
- Submerge slides in fresh 100% ethanol 1 min RT
- Submerge slides in fresh 100% ethanol 1 min RT
- Submerge slides in fresh 100% ethanol 1 min RT
- Submerge slides in fresh Pro-Par 5 min RT
- Submerge slides in fresh Pro-Par 5 min RT
- Submerge slides in fresh Pro-Par 5 min RT
- Mount slides by adding 2-4 drops of mounting media to each slide, followed by application of a cover glass. Remove bubbles from tissue by applying pressure to cover glass
- Place slides flat in a dry, dark space to air dry at RT overnight
- Assess staining with a bright-field microscope
While slides are air drying:
- Discard chromogen detection and counterstaining reagents