In situ immunoglobulin G (IgG) detection in formalin-fixed, paraffin-embedded (FFPE) pig tissues

Jayne E Wiarda, Crystal Loving

Published: 2023-08-12 DOI: 10.17504/protocols.io.e6nvwdjxzlmk/v1
AI 解读
浏览 1065

Abstract

An immunohistochemistry (IHC) staining protocol for in situ identification of IgG in pig tissue.

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.

Attachments

IHC_IgG_Pig_ManualAssay.pdf

Steps

Baking

1.

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

2.

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

3.

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

4.

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

5.

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

6.

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

7.

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

8.

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

9.

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

10.

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

11.

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

推荐阅读

Nature Protocols
Protocols IO
Current Protocols

An operant social self-administration and choice model in mice

Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging

Isotropic imaging across spatial scales with axially swept light-sheet microscopy

Snapshot photoacoustic topography through an ergodic relay of optical absorption in vivo

Design and fabrication of wearable electronic textiles using twisted fiber-based threads

Algorithmic assessment of cellular senescence in experimental and clinical specimens

Creating custom synthetic genomes inEscherichia coliwith REXER and GENESIS

High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation

Designer DNA nanostructures for viral inhibition

Reproducible strategy for excisional skin-wound-healing studies in mice

查看全部

扫码咨询