Mouse IHC

(1)

Challenges in the immunohistological

analysis of mouse tissue and cells

(2)

What we do

 _{Provide histology and IHC services to}

investigators at FHCRC, our collaborators, other institutions, and companies

 _{Majority of our samples are mouse, human,}

dog

(3)

(4)

Great IHC starts at Necropsy!

 _{Make sure you have well established} necropsy protocol

 _{The more consistent, the better the staining} results

(5)

(6)

Important Points at Necropsy

 _{Autolysis can cause background}  _{Do not let tissue dry out}

 Edge effect of IHC

 _{Get into fixative or OCT immediately}

(7)

To Fix or Not to Fix

 _{To fix}

 _{Better morphology}

 _{Prevent antigen elution or degradation}  Preserve position of antigen

 Must standardize fixation protocol!

 _{Not to fix}

 Antibody will not detect fixed antigen  _{Antigen retrieval does not work}

 _{If you are not sure, try both}

 _{We recommend preparing initial samples in both}

(8)

Mouse Spleen

OCT Embedded

CD4

(9)

Need to fix!

 _Cytokines

 _{Should be localized in the cytoplasm}

 Without fixative, these molecules can wash out

of the cell

 _{Negative staining}

 Cell not making cytokine?  Antigen washed out of cell?

 _{Estrogen Receptor}

 Location of ER is diagnostically important

 Elution of receptor from nucleus to cytoplasm

(10)

What to Fix With

 _{No single fixative ideal for all} markers, antibodies, or

applications

 10% Neutral Buffered

Formalin (NBF) suitable for 95% of applications

 _{Know the limitations and} range of fixative

 _{Fixative preparation and} protocol must remain

(11)

How Long to Fix

Under fixation

is a much greater

problem than

(12)

Why?

 _{We can use antigen retrieval techniques to} detect or “rescue” most antigens

 _{If you underfix in formalin and then process} through alcohol, you fix antigen 2 different ways

(13)

Cytokerati n WSS

Formalin fixed less

than 12 hours Formalin fixed 5 days

Ki67

(14)

How Long to Fix

 _{Know the penetration and fixation rate of your} fixative

 _{Formalin is a paradox}

 Quick to penetrate

 0.5mm per hour at room temperature

 Slow to fix

 requires days to completely cross-link

 _{Data from tissue fixed from 30 minutes to 1 year}

 72 hours sufficient for reproducible results

 Less time results in wide rage of staining variability

 No difference in staining from fixing 3 days to 12

days

(15)

Frozen Tissue

 _{Do not use tissue frozen directly in liquid} nitrogen!

 Morphology is destroyed

 _{Get a good OCT procedure to preserve tissue} architecture

(16)

(17)

IHC Controls

 _Tissue/cells

 Need a good positive and negative

 _Reagent

 When trying new technique

 _{Negative Control}

(18)

Positive Control



Indicate that proper staining technique

was achieved



Use the same control over time

 Follow staining consistency over time  Familiarity with pattern



As close to experimental as possible

 Size and shape

 Fixation and processing

 _{Antigen intensity and distribution}

(19)

(20)

Positive Control



Not the same as “It should be in this

tissue”

(21)

Isotype Control



Irrelevant immunoglobulin from the same

species and subtype as primary



Used to determine non-specific binding of

primary antibody



Must be concentration matched with the

primary and use the same staining

procedure

 Primary antibodies in research are often used

(22)

Heart stained with Smooth

Muscle Actin

(23)

Isotype Control

 _{“Secondary only” or “no primary” helpful} during work-up and troubleshooting

(24)

Primary antibodies

How do you pick your antibody?

1.

It’s been made against the species of

interest or cross-reacts

2.

Works in your system – unfixed, frozen,

or formalin fixed, paraffin embedded

3.

Robust and consistent staining

(25)

“Well, they got it to work ...

A) in this paper!”

B) at that lab!”

C) with this fixative!”

“But it worked

(26)

Primary antibodies

Where do I find them?

1. Search Engines

2. List Serv/ Histonet

3. Published papers

4. Researchers web pages

5. Company Catalog/Website

(27)

Search Engines

 _{The Antibody Resource Page}

(www.antibodyresource.com)

 _{Biocompare (www.biocompare.com)}

 _{Lab Velocity (researchlink.labvelocity.com)}

 _{Linscott's Directory}

(www.linscottsdirectory.com)

(28)

Research Group Web Sites

Antibody clone /cat# Source species reactivity Notes

Lymphoid antigens

CD3e Dako rabbit all T cells R,H CD3e CD3-12 * Novocastra rat all T cells H CD3z 6B10.2 SCB mo IgG1 all T cells Notes R, H CD22 2D6 SBA rat all B cells

CD28 sc-1624 SCB goat T cells R, H CD30 mCD30.1 * BD Pharmingen hamster activated cells

CD34 RAM34 * BD Pharmingen rat

precursors,

endothelia Notes CD40 sc-1731 SCB goat

Dendritic cells, B

cells H CD45R / B220 RA3/6B2 * SBA rat all B cells, NK

CD45RC C455.1F SBA rat all B cells, NK

CD79b / B29 HM79-12 SBA rat all B cells Notes CD138 Syndecan 281-2 BD Pharmingen rat plasma cells, epithelia

Pax5 24 BD Pharmingen mo IgG1 all B cells R, H

(29)

Companies

 _{Look for antibodies made against mouse or}

that cross react

 _{Make sure they have been tested in}

Immunohistochemistry!

 _{Look for guarantees}

 _{Talk to technical services!}

(30)

Choice of secondary



Some secondaries have higher

affinity than others



Goat anti-rat vs. Rabbit anti-rat



Consider Fab’2 fragments



Reduce

nonspecific binding to Fc

(31)

Choice of secondary



Choose secondary with limited

cross-reactivity

 For example: Jackson ImmunoResearch Lab makes

a Goat anti-Mouse IgG (115-005-003)

 Or you can order the Goat anti-Mouse IgG (min X

Rat, Hu, Bov, Hrs, Rb sr Prot) (115-005-166)



May have diminished epitope recognition

(32)

Beware of “Universal” Secondaries!!



These are great if you are staining human

tissue.



They contain secondaries that recognize

antibodies made in species including

Mouse, Rabbit, and Goat primary

antibodies.



Could be a huge problem if staining

(33)

Amplification

(34)

(35)

Spleen Stained with Ki67

HRP-anti Rabbit Ki67 1:20

Avidin-Biotin Ki67 1:100

Polymer Ki67 1:200

(36)

(37)

Protein or Serum Blocking

 _Purpose:

 To block non-specific protein-protein

interaction

 Fc receptor binding

 Inhibit secondary from binding to tissue

(38)

Protein Block

 _Contains:

 Buffer (TBS)

 Proteins such as BSA (bovine serum albumin)

or casein

 Serum from species that secondary was made

in

 Serum from species of tissue you are staining

 EXCEPT: When primary antibody is also made

(39)

Protein or Serum Blocking



Example #1:

 Tissue: Mouse Lymph Node  Primary: Rat anti-Mouse CD3

 Secondary: Biotinylated Goat anti-Rat



Contains:

 Tris Buffered Saline  1% BSA

(40)

Protein or Serum Blocking



Example #2:

 Tissue: Mouse Spleen

 Primary: Mouse anti-Human SMA

 Secondary: Biotinylated Goat anti-Mouse



Contains:

 Tris Buffered Saline  1% BSA

 15% Serum  5% _MouseGoat Serum

(41)

Bleomycin-Damaged Mouse Lung Goat anti-TNF-alpha

Biotinylated Donkey anti-Goat

15% Donkey 5% Mouse

(42)

Fc Receptors (FcR)

 _{What tissues and cells}

 Macrophages, neutrophils, eosinophils, Dendritic

cells, platelets, Langerhans cells, tissue macrophages, B cells, T cells, Mast cells, monocytes, magakaryocytes

 Endothelial cells, epithelial cells, mesangial cells  Constitutive or activated

 _{Can be released as soluble receptor}

 _{Bacterial FcR}

 _{Virally encoded FcR}

(43)

(44)

Avidin/Biotin Background



Endogenous biotin, biotin receptors, or

avidin binding sites present in tissue



Kidney, Liver, GI Tract, Spleen, Brain,

Breast, Adipose Tissue, Lymphoid Tissue



Higher in antigen retrieved or unfixed

samples



Can be blocked

 _Avidin

(45)

Using Mouse Monoclonal Antibodies on

Mouse Tissue

 _{Most monoclonal antibodies are still made in} mouse

(46)

B B

B

A

(47)

Mouse anti-Human

SMA with

Biotinylated Goat

anti-Mouse

Mouse anti-Human

IgG2a with

Biotinylated Goat

anti-Mouse

(48)

Solution #1

Don’t use a mouse monoclonal

 _{Can you use a different primary antibody?}  _{One not made in mouse}

EpCAM

(49)

Solution #2

Use a directly labeled primary

antibody!

 _{Won’t work if you need to amplify the signal}

 Can amplify using antibodies directed against

label

 _{Techniques to directly label an antibody}

 Labeling antibody can be harsh and time

(50)

Solution #3

Block Endogenous Mouse

antibodies

HAM Block

•

Use Horse anti-Mouse to bind to all

endogenous antibody

•

Make sure use unlabeled HAM antibody

Commercial Kits

•

Chemicon Mouse-To-Mouse

•

Vector M.O.M.

(51)

A

(52)

Solution #4

Complex the primary antibody with the

biotinylated secondary in a tube first

•

Block free secondary with mouse serum

•

Then apply to tissue

•

Commercial Kit

(53)

B

(54)

A

(55)

Mouse Heart stained with ARK Kit

(56)

Solution 5

Use isotype-specific anti-mouse

secondary antibodies

 _{Each IgG isotype only makes up 20-25% of}

the serum

 _{Most mouse monoclonal antibodies are IgG1}

 _{http://icg.cpmc.columbia.edu/cattoretti/Protocol/}

(57)

IgG2b _IgG1

IgG1

B B

Anti-mouse IgG1

B A

A

(58)

Mouse Heart

Mouse anti-SMA

(59)



Example: Human hematopoetic and

mesenchymal stem cells injected into mouse.



Question: Where did the human cells go?



Problem: Only want to detect Human immune

and mesenchymal cells. Not mouse.

(60)



Solution:

 _{Use anti-human CD45 to detect immune cells and}

anti-cow vimentin to detect mesenchymal cells

 Both antibodies do not react with mouse antigen  _{Use mouse-on-mouse system to avoid}

background



Result:



Detected human cells in most tissues of

(61)

Spleen

CD45

SMA IgG

(62)

The

Experimental Histopathology

Team

Over 130 Years of Histology

(63)

FHCRC Experimental Histopathology Shared

Resources

Lab Contact Info

1100 Fairview Ave N. DE-360

Seattle WA 98109-1024 206-667-6166

Julie Randolph-Habecker, Ph.D.

 _{[email protected]}