Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies

Abstract

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.

Introduction

The research on routinely processed tissue (formalin-fixed, paraffin-embedded: FFPE) via immunostaining has exploded after the introduction of antigen retrieval and the availability of reproducible primary antibodies (Abs), nowadays in monoclonal or recombinant form, raised in rabbits, mice, rat, etc. The detection of unconjugated primary Abs by anti-immunoglobulin (Ig) secondary Abs has provided a cheap, versatile, and robust method which is at the basis of histopathology immunodiagnostics and research. But this is only true for human FFPE tissue, where the secondary anti-mouse reagents are preadsorbed so that they do not detect endogenous human Ig.

When applying primary Abs raised in mice to FFPE mouse tissue, inevitably secondary Abs do detect endogenous Ig, in plasma cells, B cell, intestinal epithelial cells, serum, interstitial space, and in every instance where endogenous Ig are present.^1–3 This makes it impossible to use a simple primary–secondary sequence when both the source of the primary Ab and the tissue are from the same host.

To overcome this limitation, various experimental and commercial solutions have been proposed.

The simplest is to use directly conjugated primary mouse Ig, typically in fluorescence, a solution which applies best to frozen mouse tissue because the antigens are lightly fixed, a broad selection of Abs and conjugates is available, and the sensitivity is superior to FFPE tissue. Haptenated mouse primary Abs coupled with horseradish peroxidase (HRP)-conjugated secondary Abs for immunohistochemical (IHC) staining may be a solution, which has not been explored thoroughly.

A solution which has been commercially made available is to somehow shield the endogenous Ig in the tissue from the detection by the secondary anti-mouse Ig, leaving only the primary Ab applied to the tissue to be detected. The application of Fab monomers of anti-mouse anti-heavy and anti-light chain Abs (H+L) before the application of the primary Ab would fit that purpose.

Alternatively, conjugated Fab fragments of anti-mouse Abs are complexed with the unconjugated primary Ab before applying to the tissue, in a sort of ad hoc conjugation of the Ab with a reporter-carrying secondary Ab.^2,3

Because of these problems, most of the IHC staining on mouse tissue relies on the ever-expanding catalog of rabbit monoclonal Abs or Abs raised in goats, hamsters, rats etc., where the secondary reagents are absorbed with mouse Ig, preventing any cross-reactivity.

While experimenting with staining with mouse primary Abs on FFPE mouse tissue,⁴ we found a simple, straightforward solution which applies to any immunostaining, fluorescent, or in IHC, using mouse Abs on FFPE mouse tissue: anti-isotype secondary Abs.

Materials and Methods

Mouse and rat tissues were leftovers of approved experiments. Three common mouse strains were analyzed: C57Bl/6N, BalbC, and FVB/N. Rat Sprague Dawley strain was used. All animal experiments from which tissue was obtained were approved by the intramural animal welfare committee for animal experimentation of Chiesi Farmaceutici under protocol number: 449/2016-PR and comply with the European Directive 2010/63 UE, Italian D.Lgs 26/2014, and the revised “Guide for the Care and Use of Laboratory Animals” (Committee for the Update of the Guide for the Care and Use of Laboratory Animals and National Research Council, 2010).

Five-µm sections from FFPE tissue on silanized slides were dewaxed and antigen retrieved as published.⁵ No blocking step was performed.⁶ Endogenous peroxidase was inactivated.⁷

Primary Abs used are listed in Table 1. Incubation with appropriately diluted mouse serum or Ab dilution buffer only did not show any difference in staining (not shown). Primary Ab or control Ig incubation time was 1 hr. The anti-mouse Ig Abs or the HRP-conjugated polymers were incubated for 30 min each, all at room temperature.

Table 1.

Primary and Secondary Antibodies.

Target	Clone	Species	Working Conc./Dilution	Cat. No.	Company	Lot	RRID:AB_
Primary antibodies
Nil	N/A	All isotypes (mouse serum Ig)	2 µg/ml	011-000-002	JIR	125643	2337187
CTNNB1	#14	Mouse IgG1	1 µg/ml	610154	Becton Dickinson (BD) Pharmingen	88001	397555
Ki-67	8D5	Mouse IgG1	1 µg/ml	SAB5300425	Merck-Sigma	1012212	10979995
Ki-67	UMAB107	Mouse IgG2a	1 µg/ml	UM800033	Origene	UM800033	2629145
TCF1/7	C5	Mouse IgG2a	1 µg/ml	sc-271453	SCBT	J1717	10649799
Host	Target	Format	Specificity	Cat. No.	Working Conc./Dilution	Company	Notes	Lot	RRID:AB_
Secondary antibodies
Gt	IgG1	Biotin	AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 1 specific	115-065-205	2 µg/ml	JIR	Whole IgG	100878	2338571
Gt	IgG2a	Biotin	AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2a specific	115-065-206	2 µg/ml	JIR	Whole IgG	104859	2338572
Gt	IgG2a	ATTO 488	Affinity Purified Goat Anti-Mouse IgG, Fcγ subclass 2a specific	610-152-041	2 µg/ml	Rockland	Whole IgG	31155	2614855
Gt	IgG2a	CF488A	Affinity Purified Goat Anti-Mouse IgG, Fcγ subclass 2a specific	SAB4600239	2 µg/ml	Sigma	Whole IgG	11C1003	NA
Gt	IgG2b	Alexa Fluor 488	AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 2b specific	115-545-207	2 µg/ml	JIR	Whole IgG	133657	2338856
Gt	IgG2b	Alexa Fluor 680	Affinity Purified Goat Anti-Mouse IgG, Fcγ subclass 2b specific	A31564	2 µg/ml	Invitrogen	Whole IgG	923450	1500893
Gt	IgG3	Alexa Fluor 647	AffiniPure Goat Anti-Mouse IgG, Fcγ subclass 3 specific	115-605-209	2 µg/ml	JIR	Whole IgG	144446	2338920
Gt	IgG1	Unconjugated	AffiniPure Fab Fragment Goat Anti-Mouse IgG1, Fcγ fragment specific	115-007-185	2 µg/ml	JIR	Fab	126091	2632498
Gt	IgG2a	Unconjugated	AffiniPure Fab Fragment Goat Anti-Mouse IgG2a, Fcγ fragment specific	115-007-186	2 µg/ml	JIR	Fab	126677	2632499
Gt	IgG2b	Unconjugated	AffiniPure Fab Fragment Goat Anti-Mouse IgG2b, Fcγ fragment specific	115-007-187	2 µg/ml	JIR	Fab	126866	2632500
Gt	IgG H+L	Unconjugated	Affinity Purified Fab fragment Goat Anti-Mouse IgG (H+L) Antibody	810-1102	2 µg/ml	Rockland	Fab	29892	218897
Rb	IgG H+L	Unconjugated	AffiPure Rabbit anti-Mouse IgG (H+L)	315-005-045	2 µg/ml	JIR	Whole IgG	113040	2340038
Rb	Mo Fcγ	Unconjugated	AffiniPure Rabbit Anti-Mouse IgG, Fcγ fragment specific	315-005-046	2 µg/ml	JIR	Whole IgG	125565	2340039
Horse	Mo H+L	HRP	ImmPRESS HRP Horse Anti-Mouse IgG PLUS Polymer Kit, Peroxidase	MP-7802	N/A	Vector Labs	—	ZG0618	2336535
Horse	Rb H+L	HRP	ImmPRESS HRP Horse Anti-Rabbit IgG PLUS Polymer Kit, Peroxidase	MP-7801	N/A	Vector Labs	—	ZF0724	2336536
Horse	Goat H+L	HRP	ImmPRESS HRP Horse Anti-Goat IgG Polymer Kit, Peroxidase	MP-7405	N/A	Vector Labs	—	ZG0923	2336526

Note that the secondary antibodies have been absorbed with immunoglobulins from multiple mammals (human, rabbit, mouse, rat, etc.) to avoid cross-reactivity. For individual antibody absorption, consult the commercial source. Abbreviations: Ig, immunoglobulins; JIR, Jackson Immuno Research; SCBT, Santa Cruz Biotechnology; Gt, goat; Rb, rabbit; HRP, horseradish peroxidase; Mo, mouse.

Polymers carrying human serum-absorbed horse anti-mouse (H+L) Ig, conjugated to HRP (Table 1), were used as suggested by the manufacturer. Whole or Fab fragments of anti-mouse H+L Ig chains or anti-isotype, raised in goat and rabbit (Table 1), were counterstained with anti-goat/sheep or anti-rabbit HRP-conjugated polymers (Table 1).

The IHC stains were developed in diaminobenzidine (K3468; Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Italy), lightly counterstained in hematoxylin, mounted, and scanned on an S60 Hamamatsu scanner (Nikon; Campi Bisenzio, Italy).

Results

Polymers carrying HRP and anti-mouse H+L Ig chains raised in horse (Table 1) applied to mouse routinely processed tissue do detect endogenous Ig, diffusely in the interstitium, in plasma cells, and in gut epithelia (Fig. 1) (Table 2). Unconjugated anti-mouse H+L Abs (Table 1), whole molecules or Fab fragments, counterstained with an HRP–Ig polymer directed at the species in which they are raised (goat, rabbit) (Table 1) also detect endogenous Ig (Fig. 1) (Table 2). The anti-goat or anti-rabbit HRP–Ig polymers (Table 1), absorbed against mouse Ig, provide a clean background (Table 2) (Supplemental Fig. 1).

Figure 1.

Immunostaining of C57Bl/6N mouse FFPE tissue (spleen, intestine, and lymph node) for (A) horse anti-mouse H+L polymer; (B) goat anti H+L mouse Ig, unconjugated; (C) rabbit anti H+L mouse Ig, unconjugated; (D) rabbit anti-mouse IgG heavy chains, unconjugated; (E) goat anti-mouse IgG1, biotin; (F) goat anti-mouse IgG2a, biotin; (G) goat anti-mouse IgG2b, Alexa488; (H) goat anti-mouse IgG3, Alexa647. (E–F) Extranodal mast cells (star) capture the staining sequence. Note the absence of plasma cell staining in the villi in D. B and E–H are counterstained with a Horse anti-Goat/Sheep HRP-conjugated polymer. C and D are counterstained with a horse anti-rabbit polymer. Developed in DAB. Scale bar = 100 µm. Abbreviations: FFPE, formalin-fixed, paraffin-embedded; Ig, immunoglobulins; HRP, horseradish peroxidase; DAB, diaminobenzidine.

Table 2.

Staining of Endogenous Mouse Ig by Anti-Mouse Antibodies.

Host	Specificity	Format	Reagent	Second Layer	Results
First layer
N/A	N/A	N/A	Nil	Horse anti-Goat-HRP	Negative staining
N/A	N/A	N/A	Nil	Horse anti-Mouse-HRP	Positive staining
Gt	Fcγ subclass 1	Biotin	Goat a Mouse IgG1—biotin	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 2a	Biotin	Goat a Mouse IgG2a—biotin	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 2a	ATTO 488	Goat Anti-Mouse IgG, Fc Subclass 2a	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 2a	CF488A	Goat Anti-Mouse IgG, Fc Subclass 2a	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 2b	Alexa Fluor 488	Goat Anti-Mouse IgG, Fc Subclass 2b	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 2b	Alexa Fluor 680	Goat Anti-Mouse IgG, Fc Subclass 2b	Horse anti-Goat-HRP	PC staining
Gt	Fcγ subclass 3	Alexa Fluor 647	Goat Anti-Mouse IgG, Fc Subclass 3	Horse anti-Goat-HRP	Negative staining
Gt	Fcγ subclass 1	Unconjugated	Goat Anti-Mouse IgG, Fc Subclass 1	Horse anti-Goat-HRP	PC staining
Gt	Fcγ subclass 2a	Unconjugated	Goat Anti-Mouse IgG, Fc Subclass 2a	Horse anti-Goat-HRP	PC staining
Gt	Fcγ subclass 2b	Unconjugated	Goat Anti-Mouse IgG, Fc Subclass 2b	Horse anti-Goat-HRP	PC staining
Gt	IgG H+L	Unconjugated	Goat Anti-Mouse IgG (H+L)	Horse anti-Goat-HRP	Positive staining
Rb	IgG H+L	Unconjugated	Rabbit Anti-Mouse IgG (H+L)	Horse anti-Rabbit-HRP	Positive staining
Rb	Mo Fcγ	Unconjugated	Rabbit Anti-Mouse IgG, Fcγ fragment	Horse anti-Rabbit-HRP	Positive staining

Abbreviations: Ig, immunoglobulins; HRP, horseradish peroxidase; Gt, goat; PC = plasma cells; Rb, rabbit; Mo, mouse.

Fluorochrome-conjugated Abs specific for mouse heavy chain isotypes IgG1, IgG2a, IgG2b, and IgG3, however, fail to detect endogenous mouse Ig (Fig. 1) while providing a clean detection of exogenous mouse Abs on mouse tissue (Fig. 2) (Table 2). Reagents from four different providers (Table 1) reacted identically (Table 2). Fab monomers of anti-isotype Igs detected plasma cells in lymph node medullary cords, but not or minimally interstitial Igs (Supplemental Fig. 2).

Figure 2.

Immunostaining of C57Bl/6N mouse formalin-fixed, paraffin-embedded tissue (spleen, intestine) with anti-isotype secondary antibodies. (A) CTNNB1 (mouse IgG1 clone #14) staining of small intestine; note the membranous staining on the enterocytes and the nuclear staining in the crypt base. Scale bar: 100 µm. Inset: a low-power view of the same specimen. Secondary Ab: (Gt a Mo IgG1-biot.). (B) TCF1/7 (mouse IgG2a clone C5) nuclear staining of T cells in the spleen. Scale bar = 100 µm. Inset: Ki-67 staining (mouse IgG2a clone UMAB107) of small intestine. Scale bar = 100 µm. Secondary Ab: (Gt a Mo IgG2a, Fab). Abbreviations: Ab, antibody; Gt, goat; Mo, mouse.

To control for the difference between anti-isotypic and antiallotypic reactivity with endogenous Ig heavy chains, a rabbit Ab for the FC, but not the light-chain-containing Fab portion of mouse IgG, was applied and counterstained with a mouse-adsorbed, anti-rabbit HRP polymer, resulting in detection of endogenous Ig, except in gut IgA plasma cells (Fig. 1) (Table 2).

Anti-isotype staining of tissue from a total of three mouse strains produced identical staining (Supplemental Fig. 3).

Anti-mouse isotype Abs, which are not absorbed against rat Ig, do cross-react with rat Ig in suspension because of sequence similarities between the Ig of two closely related species; we tested whether anti-mouse isotypes would react with FFPE rat tissue and they do not (not shown).

Anti-mouse H+L Fab fragment block at 10 or 100 µg/ml did not prevent unwanted endogenous Ig staining (Supplemental Fig. 4).

Discussion

A very simple, cheap, and very efficient solution to the problem of cross-reactivity with endogenous Ig, when using mouse on mouse staining, is the use of anti-isotype secondary Abs on FFPE tissue sections. Not only the diffuse Ig-containing serum is not detected but also the dense Ig deposits within the plasma cell cytoplasm are negative.

Although limited in the variety of anti-isotype Ab suppliers and in examples of mouse tissue stainings, we believe that these findings, if confirmed by others, will broaden the use of mouse Abs on mouse FFPE experimental tissue.

Several factors, alone or in synergy, may produce this result.

Signal Dilution

The detection of only one of the two types of Ig chains may theoretically halve the reactivity when using anti-isotypes. In addition, while the light chains are common to all isotypes, the detection of isotypes further reduces the amount of detection, to the extent to which that isotype is represented among all the serum Ig. This may amount to a factor from a third to a fourth, depending on the mouse strain,^8,9 to be added to the halving previously mentioned. In organs where IgA are the predominant species, both in epithelia and plasma cells, avoiding detecting the light chains is a big factor. The failure to detect the cytoplasm of IgG plasma cells in indirect IHC, however (see below), suggests that additional factors may be involved.

Conformational Epitopes

The epitope composing the isotype specificity of murine Ig may be conformational in nature¹⁰ and may be destroyed by the FFPE processing. The Fc receptor, to name one, undergoes a similar inactivation upon tissue processing⁶ and several other conformational epitopes as well. However, the secondary Abs are polyclonal; thus, some Ig clones reacting with the denatured epitope may exist. In IgG-containing spleen sections, robust signal amplification is required to detect those minute amounts of FFPE-proof Abs.¹¹ It is useful to remember to this point that the efforts to raise polyclonal Abs against denatured human antigens for FFPE use failed repeatedly at the beginning of the IHC era and this may be the case for anti-isotypes.

Curiously, polyclonal Abs against both the heavy and light Ig chains, which by definition is a complex conformation of four separate proteins, two by two identical, are unfortunately very efficient at detecting denatured endogenous Ig on FFPE mouse tissue.

Preabsorption

All the reagents used have been preabsorbed against the other mouse isotypes (and human Igs). This procedure may have removed from the polyclonal Ab pool the Ig species directed against the epitopes, FFPE processing dependent or surviving, which are shared among diverse isotypes. The ones left over detect a conformational epitope(s) on the native Ig, which is destroyed upon fixation and embedding, resulting in the selective detection of the exogenous Ig applied to the section. As a confirmation of the hypothesis, an Ab raised against the mouse IgG heavy chains, not absorbed to other mouse Ig, does detect endogenous Ig in FFPE material.

Ab Format and Staining Conditions

Fluorochrome- or biotin-conjugated Abs failed to detect endogenous mouse Ig in plasma cells and in the interstitium, while Fab monomers labeled plasma cells only. Several factors may have to be taken into account for this differential reactivity. Fluorochrome or biotin conjugation adds up to the Ig molecular weight and steric hindrance, creating a bulkier product. If we add to this a short incubation time, 30 min, tailored to mimic the usual staining conditions for secondary Abs, but shortening the time to penetrate into the FFPE tissue, the combination may produce the peculiar differential reactivity we have described. To this “physical” effect, one may add the proprietary immunization protocol and affinity purification columns of the producers, which may subtly play in the recognition of soluble circulating Igs versus hypoglycosylated intracellular Ig in plasma cells, recognizing the target in an unconventional environment such as the FFPE material.

No matter what causes this very distinct unreactivity of anti-mouse Ig isotypes for FFPE mouse Ig, this effect is exploitable to expand the use of mouse Abs on mouse tissue.