CHAPTER FIFTEEN

Preparation of Formalin-fixedParaffin-embedded Tissue forImmunohistochemistryKirstie Canene-Adams1Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA1Corresponding author: e-mail address: kirstieadams26@yahoo.com

Contents

1.

MetISShttp

Theory

hods in Enzymology, Volume 533 # 2013 Elsevier Inc.N 0076-6879 All rights reserved.://dx.doi.org/10.1016/B978-0-12-420067-8.00015-5

226

2. Equipment 227 3. Materials 227

3.1

Solutions & buffers 227 4. Protocol 228

4.1

Preparation 228 4.2 Duration 228 4.3 Caution 228

5.

Step 1 Fixation of Tissue 229 5.1 Overview 229 5.2 Duration 229 5.3 Tip 229 5.4 Tip 229 5.5 Tip 229 5.6 Tip 229 5.7 Tip 230

6.

Step 2 Tissue Processing and Embedding 230 6.1 Overview 230 6.2 Duration 230 6.3 Tip 231 6.4 Tip 231 6.5 Tip 231

7.

Step 3 Tissue Sectioning 232 7.1 Overview 232 7.2 Duration 232 7.3 Tip 232 7.4 Tip 232 7.5 Tip 232 7.6 Tip 232

225

226 Kirstie Canene-Adams

7.7

Tip 233 References 233

Abstract

The purpose of this protocol is to take any biopsy or whole organ tissue from animals orhuman, formalin-fix the specimen to preserve the current state of the tissue, and embedit into a paraffin block and for future immunohistochemistry experiments (If you intendto fix cells, check the alternative protocols: Preparation of Cells for Microscopy usingCytospin, Preparation of Cells for Microscopy using Chamber Slides and Coverslips,or Preparation of Cells for Microscopy using ‘Cell Blocks’).

1. THEORY

Fixation: There are four general procedures for tissue fixation; snap

freezing, OCT media embedding, RNA stabilizing reagent, or fixative. This

protocol will focus on formalin fixation since it is the most common. There

are multiple solutions that can be used for fixation of tissues, including Bouin’s

Fixative, paraformaldehyde, and neutral-buffered formalin. Tissue is fixed by

cross-linkages between the lysine residues of proteins. The primary purpose of

a fixative is to stop the progress of proteolytic enzymes in the tissue that will

digest and damage the sample if not inhibited. Fixatives also act to inhibit

microorganism growth and maintain or improve a tissue’s strength and stabil-

ity to preserve the morphology of the sample as it continues through the

processing stages. There are a number of factors that affect fixation, such as

penetration of the fixative into the tissue, volume of the fixative and the tissue

sample, temperature, concentration of fixative, and the time allowed for

fixation. Fixation is best carried out at a neutral pH, in the range of 6–8;

commercially available formalin is buffered with phosphate at a pH of 7.

Processing and embedding: After a tissue has been fixed, it must be processed

so that it can ultimately be cut into thin sections suitable for microscopy.

Initially, specimens are dehydrated to remove water and then cleared, the

process by which the solution used to dehydrate the tissues is replaced with

a new solution that is miscible with paraffin, since wet-fixed tissues cannot

be directly penetrated with paraffin. The tissue sample is the then penetrated

with the embedding agent, which is most commonly paraffin. Processing

and embedding are frequently automated using an instrument that makes

the tissue samples progress from stage to stage on a standard time schedule.

Specimens that have been formalin-fixed and embedded in paraffin can be

later sectioned and stained with hematoxylin and eosin (H&E). Hematox-

ylin stains nuclei blue and eosin stains the cytoplasm pink. Formalin-fixed

paraffin-embedded tissue (FFPE) offers the best morphological

227Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry

characteristics of tissue specimens, and FFPE sections can be used for immu-

nohistochemical analyses of protein expression.

2. EQUIPMENT

Tissue fixation cassettes

Beaker or container with lid (to submerge cassettes with enough room to

fully cover them with liquid at a ratio of 10:1 fixative volume to tissue

volume)

Automated Tissue Processor, if available

Paraffin warmer

Paraffin embedding mold

Water bath (35–40 �C)Microtome

Oven (65 �C)Razor blades

Forceps

Cutting board or surface (must be clean)

3. MATERIALS

Formalin solution, neutral buffered, 10%

Sodium chloride (NaCl)

Potassium chloride (KCl)

Sodium phosphate dibasic (Na2HPO4)

Potassium phosphate monobasic (KH2PO4)

Ethanol (70%, 95%, and 100%)

Xylene

Paraffin

3.1. Solutions & buffersStep 1 1� PBS

Component

Final concentration Amount

NaCl

137 mM 8 g

KCl

2.7 mM 0.2 g

Na2HPO4

10 mM 1.44 g

KH2PO4

1.76 mM 0.24 g

Dissolve in 800-ml ultrapure water. Adjust pH to 7.4 using HCl. Add water to a final volume of 1 l.Sterilize by autoclaving

228 Kirstie Canene-Adams

4. PROTOCOL

4.1. Preparation

Figure

Collect human tissue sample or laboratory animal tissues according to lab-

oratory protocol and institutional guidelines.

4.2. Duration

Preparation

15.1 Flowchar

Variable, depends on the time it will take for tissue collection

Protocol

48 h (formalin fixation)

�24 h (processing)

�12 h (embedding)

�4–8 h (sectioning)

Embedding and sectioning times will vary greatly depending on the

number of tissues and the skill of the scientist.

4.3. CautionWork in a well-ventilated area and wear gloves when working with fixatives. Forma-

lin contains formaldehyde, a severe eye, respiratory and skin irritant that is toxic by

ingestion and inhalation. It is also considered to be carcinogenic and corrosive. Consult

MSDS for precautions and first aid treatment.

See Fig. 15.1 for the flowchart of the complete protocol.

t of the complete protocol, including preparation.

229Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry

5. STEP 1 FIXATION OF TISSUE

5.1. Overview

The purpose of this step is to preserve the tissues with formalin so that they

remain as biologically similar to when they were removed as possible.

5.2. Duration48 h

1.1 As soon as you harvest a tissue, cut it to a thickness no greater than

3–4 mmusing a sharp, clean razor blade and place specimen into a tissue

fixation cassette.

1.2 Immediately put the cassette containing the tissue into a beaker con-

taining formalin at a ratio of 10:1 fixative to tissue volume. Cover

the beaker with two layers of foil. Alternatively use a plastic container

with a lid.

1.3 Let samples sit for 48 h in formalin.

1.4 Pour formalin into a waste container for proper disposal, rinse tissues in

cold water for about 5 min, and place tissues in PBS. Store at 4 �C until

ready for processing.

5.3. TipThe time between removal of tissues to fixation is important, and the faster you can put

the tissue into formalin, the better. Ideally, specimens should be placed in formalin

within 5 min from harvesting.

5.4. TipSection the tissues thinly (3–4 mm) when putting into cassettes so that the formalin

can penetrate into the tissue, as a thin section will fix more rapidly than a thick section.

5.5. TipAlways be sure to label your cassette well using a pencil!

5.6. TipIf the tissues will not be processed for weeks, or for long-term storage, place them in

70% ethanol instead of PBS.

Figure 15.2 Flowchart of Step 1.

230 Kirstie Canene-Adams

5.7. TipTell the histologist what you fixed your tissues in, as this is vital for the processing/

embedding process of the tissues.

See Fig. 15.2 for the flowchart of Step 1.

6. STEP 2 TISSUE PROCESSING AND EMBEDDING

6.1. Overview

The purpose of this step is to dehydrate the tissue and embed it in a paraffin

block.Most often this is done overnight using an automated tissue processor,

but this protocol will use the same principles and can be performed without

the machine.

6.2. Duration3–4 h or overnight (for processing, using an automated tissue processor) a

few hours (for paraffin to set up)

2.1 Place the tissues, while still in the cassettes, into 70% ethanol for

20 min.

2.2 Transfer the tissues into 95% ethanol for 20 min. Repeat incubation in

fresh 95% ethanol.

2.3 Transfer the tissues into 100% ethanol for 20 min. Repeat incubation in

fresh 100% ethanol.

2.4 ‘Clear’ the tissues by placing the tissues into a xylene bath for 20 min to

remove the ethanol. Repeat incubation in fresh xylene.

231Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry

2.5 Open the cassettes and embed the tissues in molten paraffin for 30 min

to replace the xylene in the tissues. Fill the mold with paraffin to make a

‘block.’

2.6 Allow paraffin to cool and harden.

6.3. TipThe incubations in ethanol and xylene can be done with the tissues still in the cassettes.

Many automated machines will do these steps for you overnight.

6.4. TipCarefully straighten and align the tissues properly in the block of paraffin.

6.5. TipBe careful with molten paraffin; it has a melting point of around 55–65 �C.

See Fig. 15.3 for the flowchart of Step 2.

Figure 15.3 Flowchart of Step 2.

232 Kirstie Canene-Adams

7. STEP 3 TISSUE SECTIONING

7.1. Overview

The purpose of this step is to make cuts or slices of the block, which contains

the tissue embedded in paraffin, and mount these sections on glass micro-

scope slides.

7.2. DurationWill depend greatly on the skill of the user

3.1 Have a water bath ready at 35–40 �C.3.2 Place the tissue block in a microtome, cryostat, or another sectioning

device you have access to, so that it is parallel to the cutting blade.

3.3 Cut the tissues into slices between 4–5 mm in thickness.

3.4 Place the cut slices of tissue onto the surface of the water.

3.5 Place a glass microscope slide under the floating tissue slice and lift the

slide up to catch the tissue section on the slide.

3.6 Place the slides in an oven at 65 �C for 10–20 min until the paraffin

starts to melt, to mount the tissue to the slide.

7.3. TipIf the block is not cutting well, the paraffin could be too warm. Place the blocks on ice to

cool them down.

7.4. TipIf the tissue sections break up when placed on the surface of the water, the temperature

of the bath is too warm.

7.5. TipAs with the paraffin blocks, it is always a good idea to have your slides labeled well!

7.6. TipIt is preferred to allow slides to air-dry rather than dip them in paraffin or oven-bake

them for long periods of time as this allows preservation of the antigens for future

immunohistochemistry (see a method on Immunohistochemistry on Freely Floating

Fixed Tissue Sections).

Figure 15.4 Flowchart of Step 3.

233Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry

7.7. TipIt is best to store blocks and stained slides at room temperature and unstained slides

in a �20 �C freezer, tightly wrapped in Parafilm or in plastic bags.

See Fig. 15.4 for the flowchart of Step 3.

REFERENCESRelated LiteratureProtocol developed in the laboratory of Angelo M. De Marzo, MD, PhD for The Tissue

MicroArray Core at Johns Hopkins School of Medicine. http://tmalab.jhmi.edu/histology.html (accessed April–May 2010).

Theory from:Webpath: The Internet Pathology Laboratory for Medical Education, Mercer University

School of Medicine, Savannah, The University of Utah Eccles Health Sciences Library.http://library.med.utah.edu/WebPath/webpath.html#MENU (accessed April–May 2010).

Referenced Protocols in Methods NavigatorPreparation of Cells for Microscopy using Cytospin.Preparation of Cells for Microscopy using Chamber Slides and Coverslips.Preparation of Cells for Microscopy using ‘Cell Blocks’.Immunohistochemistry on Freely Floating Fixed Tissue Sections.