Fixatives (updated)

acetone/alcohol fixative type

non-additive, coagulant

alcohol/acetone advantages (2)

• fast penetration

• preserved enzymes at cold temperatures

alcohol/acetone disadvantages (2)

• loss of lipids

• bad for electron microscopy

alcohol/acetone preferred use (3)

• glycogen preservation

• cytology

• urate demonstration

acetic acid fixative type

non-additive, coagulant (nucleic acids)/non-coagulant (cytoplasm)

acetic acid preferred use

counteract the tissue shrinkage in fixatives

acetic acid advantage

• fast penetration

acetic acid disadvantage (2)

• lyses red blood cells

• bad for electron microscopy

Formaldehyde preferred use

to make different formalin solutions

formaldehyde fixative type

Additive, non-coagulant

formaldehyde advantages (4)

• cheap

• stable

• flexible with staining

• enzyme stains if solution is cold

formaldehyde disadvantage (4)

• gradual loss of lipids over time

• penetrates quickly but fixes slowly

• hardens tissue more than any other fixative other than alcohol and acetone

• formation of formalin pigment

10% aqueous formalin disadvantage (2)

• formalin pigment

• hypotonic

10% aqueous formalin fixative type

additive, non-coagulant

10% neutralized formalin ingredients (3)

• formaldehyde

• DI water

• calcium/magnesium carbonate

10% neutralized formalin fixative type

additive, non-coagulant

10% neutralized formalin disadvantage (2)

• acidic after withdrawal from a storage bottle

• formalin pigment

10% NBF ingredients (4)

• formaldehyde

• DI water

• sodium phosphate, monobasic and dibasic

10% NBF preferred use

routine fixative

10% NBF fixative type

Additive, Non-coagulant

10% NBF advantages (2)

• stable pH

• no formalin pigment, like other formaldehyde fixatives

10% NBF disadvantage

hypotonic

alcoholic formalin ingredients (3)

• ethanol

• DI water

• formaldehyde

alcoholic formalin preferred use (2)

• fixes and dehydrates at the same time

• trichrome stains

alcoholic formalin advantage

• better than NBF in preserving carbohydrates

alcoholic formalin fixative type

additive, coagulant

formalin ammonium bromide type of fixative

additive, non-coagulant

formalin ammonium bromide ingredients (3)

• formaldehyde

• ammonium bromide

• DI water

formalin ammonium bromide preferred use (2)

• fixation of CNS

• cajal gold stain

formalin ammonium bromide disadvantages (5)

• not good for IHC, H&E, or molecular studies

• gives positive fuelgen reaction due to hydrolysis

• lyses red blood cells

10% formalin saline type of fixative

additive, non-coagulant

10% formalin saline ingredients (3)

• water

• formaldehdye

• sodium chloride

10% formalin saline preferred use (2)

• preserve salinity of delicate tissues (neuro and embryo)

• storage and transport

10% formalin saline advantages (2)

• isotonic

• good for Masson’s technique

10% formalin saline disadvantage

not buffered, may produce formalin pigment

calcium formalin type of fixative

additive, non-coagulant

calcium formalion ingredients (3)

• DI water

• formaldehyde

• calcium chloride

calcium formalin preferred use (2)

preservation of phospholipids and myelin sheaths

calcium formalin notes (2)

• changes phospholipid solubility

• used for Baker’s acid hematein test

modified millonig formalin ingredients (4)

• formaldehyde

• DI water

• sodium phosphate monobasic

• sodium hydroxide

modified millonig formalin type of fixative

additive, non-coagulant

modified millonig formalin preferred use

electron microscopy on stored tissue

modified millonig formalin advantages (3)

• better for ultrastructure preservation

• isotonic

• buffered

modiified millonig formalin disadvantge

• paraffin sectioning is more difficult

acetate formalin ingredients (3)

• formaldehyde

• DI water

• sodium acetate

acetate formalin type of fixative

additive, non-coagulant

acetate formalin preferred use

an alternative when phosphate salts are not available

acetate formalin advantage

buffered

acetate formlin disadvantage

pseudocalcification will happen if calcium acetate is used in solution

glutareldehyde fixative type

additive, non-coagulant

glutaraldehyde preferred use

• electron microscopy

glutaraldehyde advantage

fixes while it penetrates

glutaraldehyde disadvantages (5)

• penetrates slowly but fixes rapidly

• breaks down when exposed to air

• overhardens if fixed for over two hours, put in buffer solution afterwards

• false positive in PAS staining

• gradual loss of lipids over time

how to prep vials of glutaraldehyde for electron microscopy

fresh reagent before use, prepped vials that were sealed under inert nitrogen

glyoxal preferred use (4)

• detects microcalcifications in breast biopsies

• alternative to formaldehyde for routine H&E

• can fix carbs OR proteins at different rates, at different pHs

• good preservation of chromatin and membranes

glyoxal fixative type

additive, non-coagulant

glyoxal advantages (2)

• faster than NBF

• less antigen retrieval needed for IHC

glyoxal diasvantages (3)

• weak staining after prolonged exposure

• bad for H pylori staining

• not as good as 10% NBF for enzyme stains

mercuric chloride fixative type

additive, coagulant

mercuric chloride preferred use

• in other fixatives as a protein coagulant

mercuric chloride advantage (2)

• leave tissue receptive to dyes

• rapid penetration

mercuric chloride disadvantage (5)

• mercury pigment

• cannot due frozen sections

• penetrates poorly

• shrinkage

• hardening

how to prevent mercury pigment

mercury pigment cannot be prevented

how to remove mercury pigment

iodine, followed by sodium thiosulfate (hypo)

osmium tetroxide fixative type

additive, non-coagulant

osmium tetroxide preferred use (2)

• secondary fixative for electron microscopy

• preserve lipids an phospholipids

osmium tetroxide advantages (2)

• preserve lipids and phospholipids from processing

• fixative and a stain

osmium tetroxide disadvantages (3)

• cytoplasm cannot be stained with eosin but rather hematoxylin

• expensive

• slow penetration

picric acid type of fixative

additive, coagulant

picric acid preferred use (2)

• glycogen fixation

• mordant for trichrome stains

picric acid advantages (3)

• fixative and a stain

• leave tissue receptive to dyes

• gives a soft texture to tissue

picric acid disadvantages (5)

• not good for nucleic acid stain, hydrolyzes

• shrinkage

• must be washed with 50% ehtanol before processing or else staining will be weak and morphology is destroyed

• explosive

• slow penetration

potassium dichromate type of fixative

additive, non-coagulant in alkaline solution, coagulant in acidic solution

potassium dichromate preferred use (3)

• preverve lipids

• preserve mitochondria

• preserve chromaffin granules to diagnose pheochromocytomas

potassium dichromate advantages (2)

• greater affinity for eosin

• soft tissue texture

potassium dichromate disadvantages (3)

• dissolves DNA

• shrinkage after processing

• chromate pigment

how to prevent chromate pigment

1% HCl with 70% ethanol for 30 min

how to remove chromate pigment

you cannot remove chromate pigment, but it can be reduced with acid alcohol

zinc salt fixative type

additive, coagulant

zinc salt preferred use (2)

• replacement for mercuric chloride

• post fixation on open processors

zinc salt advantages (2)

• enhanced staining

• less antigen retrieval needed for IHC

zinc salt disadvantages

• zinc precipitate

• slow penetration

what causes zinc precipitate (6)

• carbonates in tap water

• phosphates in buffered formalin

• alcohol

• heat

• pressure

• vaccuum

how to remove zinc precipitate

5-20% acetic acid solution

affects of zinc precipitate (3)

• reduce processor alcohol concentrations

• can clog processor

• difficult microtomy

B5 ingredients

B5 type of fixative

B5 preferred use

• lymph node and bone marrow (w/ zinc salts)

B5 advantages

• good antigen preservation for IHC (w/ zinc salts)

B5 disadvantages