M cells are located on Peyers patches, dome area - you did not say what
species you are working with?  Publications cited give human, mouse and
rabbit species results.  

UEA1 from Vector is superb. - we do frozen sections with UEA1-FITC, but
autofluorescence with FFPE will be a problem  

It is a simple immunostaining protocol but TBS should be used or the Lectin
buffer.  There is a fabulous book titled Lectin Histochemistry, Brooks et
al from Springer Verlag as lectins are plant proteins that bind to the
fucose oligosaccharide sugar counterpart on human tissues/cells.  M cells
can be detected this way, even with IHC.

If working with human tissue, positive control is striated muscle.



This is basically the protocol from that book

Deparaffinize
Trypsin digestion for 10 min at 37C
rinse sections in tap water (you can use buffer)
Block endogenous peroxidase
Rinse
Ulex europeus I (UEA I) diluted at 10 ug/ml
Negative control is the inhibiting sugar + UEAI at same concentration
overnight at 4C, then apply to section.  You need to have the UEAI bind to
its inhibition sugar so it DOES NOT bind the the fucose counterpart on
tissue.  Vector catalog has the concentration of sugar, 100 mM but we
increase to 300mM with overnight incubation rather than 1 hour at RT.
Often the negative control, inhibition binding is only 80% - we had totally
negative control with overnight to improve the partial negative control
problem. 

Incubate no more than 60 minutes with UEAI, or you will get binding to
other tissue components.  30 minutes is time we use.  

Rinse with TBS

Incubate with rabbit antiUEAI-HRP diluted 1:200 (Use Vector antibody) in
TBS for 1 hour

Rinse

Then DAB or chromogen of choice. 

rinse, hematoxylin counterstain, dehydrate, mount coverslip. 

Do not use serums to block, lectins bind to serums - use BSA instead, high
quality i.e. Jackson Immunoresearch protease/immunoglobulin free was
agreeable with Vector Tech services. 

We fill the gut or orient the sample so Peyers patches are cut in
midsaggital plane i.e. longitudinal.  

Publications that are must reads for technics, etc. 

Lectin binding defines and differentiates M cells in mouse small intestine
and caecum, Clark MA et al, Histochem Cell biol 1995 104(2):161-8.  

Infection and Immunity.  Human Intestinal M cells display the Sialyl Lewis
A antigen. Giannasca PJ et al.  67(2):946-952, 1999

J Histochem Cytochem  Glyconconjugate expression defines the origin and
differentiation pathway of Intestinal M cells.  Gebert A, Posselt W.
45(10):1341-1350, 1997.  

 

 


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu