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.

The normal and fibrotic mouse lung classified by spatial proteomic analysis

“…Limitations of the study: We employed a limited number of antibodies, compared to the potentiality of the MILAN technique (over 100); this because there is a variety of reagents to choose from which is more limited than what is available for human FFPE tissue, particularly in terms of species origins of the antibodies. Being the MILAN technique based on multiple (3 or 4) unconjugated antibodies per round, we partially overcome these limitations by using mouse antibodies on mouse tissue, background free 5 . It has been shown previously however, that the discriminating power of the dimensionality reduction algorithms is so high, that even with a reduced number of diagnostic parameters, the cell types of interests can still be identified 22 and this is the case for the present work.…”

“…Lung samples were processed as per the Milan (Multiple Iterative Labeling by Antibody Neodeposition) protocol 6 modified for mouse FFPE tissue staining. The protocol consists in the cyclic application of primary antibodies (Table 1 ) raised in multiple species, including mouse 5 , applied on the same section, nuclear staining with DAPI, autofluorescence subtraction 28 . For each round, the stained slides were scanned and saved as digital images using a multichannel fluorescence acquisition instrument (NanoZoomer S60, Hamamatsu, Japan).…”

“…All kinds of diagnostic and therapeutic approaches used on humans have been miniaturized and applied to mice 4 , including histopathologic and phenotypic examination. The problem of using antibody-based high-plex methods on mouse tissue when using primary antibodies raised in mice is the cross reactivity with endogenous immunoglobulins: we have overcome this problem by using anti-isotype secondary Abs 5 on FFPE mouse tissue, therefore we have broadened the antibody portfolio which can be used on mouse histopathology.…”

The Normal and Fibrotic Mouse Lung in Situ Classified by High Dimensional Single Cell Analysis on Routinely Processed Tissue

“…Limitations of the study: We employed a limited number of antibodies, compared to the potentiality of the MILAN technique (over 100); this because there is a variety of reagents to choose from which is more limited than what is available for human FFPE tissue, particularly in terms of species origins of the antibodies. Being the MILAN technique based on multiple (3 or 4) unconjugated antibodies per round, we partially overcome these limitations by using mouse antibodies on mouse tissue, background free 12 . It has been shown previously however, that the discriminating power of the dimensionality reduction algorithms is so high, that even with a reduced number of diagnostic parameters, the cell types of interests can still be identi ed 27 and this is the case for the present work.…”

“…Lung samples were processed as per the Milan (Multiple Iterative Labeling by Antibody Neodeposition) protocol 13 modi ed for mouse FFPE tissue staining. The protocol consists in the cyclic application of primary antibodies (Table 1) raised in multiple species, including mouse 12 , applied on the same section, nuclear staining with DAPI, auto uorescence subtraction 9 . For each round, the stained slides were scanned and saved as digital images using a multichannel uorescence acquisition instrument (NanoZoomer S60, Hamamatsu, Japan).…”