Immunohistochemistry - Paraffin section (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Mark Cadena; Ryan Trevena; Kyle Wegner; Hannah Ruetten; Anne Turco; Diya Joseph; Adam Gottschalk; Lisa Abler; Chad Vezina

Keywords

[‘immunohistochemistry’, ‘immunofluorescence’, ‘immunofluorescent’, ‘human’, ‘mouse’, ‘fetal mouse’]

Subjects

[‘Molecular biology’, ‘Immunological techniques’]

Release Date

2018-05-11

Abstract

Describes immunohistochemical staining of paraffin wax-embedded tissues. Protocol has been validated for fetal and adult stages in mouse and human tissues using fluorescent and chromogenic visualization techniques.

Reagents

” Blocking reagent (Roche #11096176001) “ Sodium citrate dihydrate (Fisher Scientific #BP327) “ Coverglass, 18x18mm square, #1 (Corning #2845-18) “ 4’,6-diamidino-2-phenylindole, dilactate (DAPI; Invitrogen #D3571) “ Dimethylformamide (DMF; Sigma #D4551) “ Dimethyl sulfoxide (DMSO; Fisher #BP231) “ Ethanol (EtOH; Pharmco-Aaper #111000-200E200G) “ Glycerol (Sigma G5516-500mL) “ Maleic acid (Sigma # M0375-500G) “ Sodium chloride (NaCl; Fisher BP358-212) “ n-propyl gallate (MP Biochemicals #102747) “ Phosphate buffered saline, Dulbecco’s, without calcium or magnesium (PBS; Fisher Scientific #SH3001304) “ Pyrex glass dish (Corning 222-R) “ Sudan Black B (VWR #ICI5208810) “ Super HT PAP pen (RPI Corp # 195505) “ Tris hydrochloride (Tris-HCl; Fisher BP153-1) “ Tween-20® (Fisher BP337-100) “ Xylenes, histological grade (Fischer Scientific #X3P1GAL)

Equipment

” 65°C oven “ Fume hood “ Analytical balance “ Decloaking Chamber™ Pro, (Biocare Medical, Concord, CA)

Procedure

Dewaxing, antigen retrieval and blocking.

Timing: ~4 h

1. Heat slides to 65°C for 5 min to remove wrinkles and increase tissue adhesion.
2. Dewax and rehydrate slides by incubating at 25°C in Xylene (3 min, repeat twice), 100% ethanol (3 min, repeat twice), 75% ethanol (3 min, repeat twice) and 50% ethanol (3 min, repeat twice).
3. Prepare a 1X working solution of citrate buffer by adding 5 mL of 100X solution to 495 mL of dH20 and place into a 8”x 8”x 2” Pyrex glass dish.
4. Place slides in glass dish and microwave on power 50 for 20 min. Allow slides to cool to room temperature before proceeding.

TROUBLESHOOTING

1. Use a kimwipe to create a dry rectangle around the tissue section.
2. Outline samples with hydrophobic pen, being careful to mark only the dry part of the slide.
3. Pipette a bead of TBSTw into the outlined region and let sit for 5 min at 25°C with no agitation so that the pap barrier can dry.
4. Tap off TBSTw and block for 1 hr at 25°C with gentle agitation in blocking buffer.
5. Dilute primary antibody/antibodies in blocking buffer, apply to slides, and incubate overnight at 4°C with gentle agitation.

Secondary antibody and coverslipping.

Timing: ~4 h

1. Wash 5 min in TBSTw at 25°C with gentle agitation (repeat 5 times).
2. Dilute flurophore-conjugated secondary antibody/antibodies in blocking buffer, apply to slides, and incubate for 60 min at 25°C in a light protected box.
3. Remove secondary antibody solution and wash with TBSTw for 5 min at 25°C in light protected box. Repeat this step 7 times.

TROUBLESHOOTING

1. Prepare DAPI working solution by combining 1 μl DAPI stock with 999 μl TBSTw, apply to slides and incubate for 5 min at 25°C in light protected box.
2. Wash with TBSTw for 5 min at 25°C in light protected box. Repeat this step 3 times.
3. Add a small bead of antifade-mounting media to slide and add cover glass (Corning #2865-18). Image ASAP.

Critical_Steps

• At step 7: Incomplete drying of hydrophobic barrier will cause tissue sections to dehydrate and will increase background staining.

Trouble_Shooting

• Step 4 Problem / Alternative goal: Antigen not detected Possible reason: Ineffective antigen retrieval Solution: To aid in Edu/BrdU retrieval, incubate in 2N HCl at 25°C for 30 min prior to antigen retrieval. Antigien retrieval can also be performed in the decloaker by immersing slides in an alkaline solution (10 mM Tris Base, pH 9.5) or a neutral solution (10 mM Tris HCl, pH 7.5) and running a two incubation cycle (cycle 1: 125°C for 5 min; cycle 2: 72°C for 0.5 min) or on the benchtop by trypsin digestion (phosphate buffered saline containing 0.1% trypsin, incubate at 37°C for 30 min) or proteinase K digestion (phosphate buffered saline containing 5 μg/mL proteinase K solution, incubate at 25°C for 5 min).

• Step 11 Problem / Alternative goal: Cell membrane counterstaining needed Solution: Add TBSTw containing Wheat Germ Agglutinin Texas Red (1:200 dilution from 1mg/ml glycerol stock, Invitrogen #W21405). Incubate for 5 min at 25°C in light protected box and tip off to remove and wash with TBSTw twice for 5 min per wash.

• Step 11 Problem / Alternative goal: High fluorescent background staining Solution: Add 0.1% Sudan Black B solution directly onto tissue section. Incubate for 25 min at 25°C. To remove excess solution, wash with TBSTw 3 times for 5 min per wash.

References

Abler LL, Keil KP, Mehta V, Joshi PS, Schmitz CT and Vezina CM (2011). A High Resolution Molecular Atlas of the Fetal Mouse Lower Urogenital Tract. Dev Dyn 240:2364-2377. PMC3177421.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium