the colonic epithelial lining is a highly regenerative tissue , with resident stem cells replenishing the tissue every few days .
this process of regeneration requires the maintenance of a proliferative stem cell compartment . over time , the newly produced daughter cells lose their proliferative potential and migrate toward the luminal surface of the intestine .
coincident with migration , progenitor cells differentiate into mature barrier - forming enterocytes or mucous producing goblet cells .
failure of this differentiation program is thought to contribute to compromised epithelial barrier such as is observed in inflammatory bowel disease and colon cancer .
detailed study of the above processes will require methodologies for isolating crypt - base and surface cell populations .
current methods for the isolation of intestinal epithelial cells include chelating agents and mechanical disassociation , resulting in the isolation of entire crypts , epithelial sheets , or single cells , dependent on experimental conditions .
these are high yield methods , yet changes in intercellular signaling have been reported under these conditions that may not represent the native environment .
laser capture microdissection allows for the collection of spatial distinct material , but achieves low molecular yield . in human colonic tissue , serial horizontal cryosectioning methods have been developed .
however , murine mucosal tissues are prohibitively small for direct appropriation of this technique . in humans , intestinal crypt lengths
( the distant between the crypt - base and surface cells ) in the colon varies between ~1001,000 m . in mice ,
the small size of murine crypts presents a challenge to the isolation of these two cell populations using existing protocols .
we now describe a low cost , high yield method for the isolation of crypt - base and surface epithelial cell population in murine colonic tissue .
tissue harvested in this manner may then be analyzed by a number of standard downstream applications , including immunofluorescence staining , rt  pcr ( real time  polymerase chain reaction ) or western blotting .
experiments used c57bl/6 mice between 10 and 12 weeks of age . all procedures using animals were reviewed and approved by the emory university institutional animal care and use committee and were performed according to national institutes of health criteria .
set up the cryostat equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade!).fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) . remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min.set the cryostat to 10 m per section and shave the oct block until it is flat .
equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade ! ) .
fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) .
remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min .
set the cryostat to 10 m per section and shave the oct block until it is flat .
prepare the razor blades : prepare 2 razor blades per sample . using a metal file , dull the sharped edge of blades .
place mice in a sealed chamber and euthanize mice by isoflurane inhalation and cervical dislocation , then spray 70% ethanol onto the abdomen and thorax . make a small incision in skin of the abdomen with scissors and then make an incision to expose the peritoneal cavity .
cut the large intestine at the anal verge and tease apart the mesentery until the colon is free .
excise a distal colon segment between 0 and 6 cm from the anus . here , the crypt depth is ~150 m ( see figure 1b ) .
stretch the tissue using dissection pins on a silicon dish in hanks buffer and let rest for 10 min at rt ( figure 1c ) .
slide a razor blade under the desired section of tissue and then pour off the hanks buffer .
pick up the razor / tissue sandwich and allow it to warm slightly by placing a finger on the top blade ( mucosal side up .
this orients the tissue so that the basal muscle layers are sectioned first . ) .
separate the sandwich and cut around the edges of the tissue segment . cut a tissue segment approximately , 0.5 cm x 1 cm . edge areas have a tendency to curl , causing the serial sections to contain many tissue layers .
note that over - cutting is better than under - cutting , so err on the side of caution .
allow the tissue to warm until the ice on top of the tissue begins to melt .
at this point quickly and carefully press the tissue into the oct block in the cryostat . remove the blade by placing a finger over the sample .
analysis of mrna or protein , place the samples in tubes with 5 crypt - base , 5 transitional , and 5 surface sections per tube .
for rna collection add 1 ml commercial isolation reagent and incubate at rt for 10 min . perform rna purification according to manufacturer s instructions .
use 5 - 10 g of total rna for cdna generation . for protein purification ,
add 0.5 ml of cold ripa lysis buffer ( plus protease inhibitors ) per 5 sections .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c . 
 
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
set up the cryostat equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade!).fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) . remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min.set the cryostat to 10 m per section and shave the oct block until it is flat . back
equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade ! ) .
fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) .
remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min .
set the cryostat to 10 m per section and shave the oct block until it is flat .
back the microtome arm away from the blade by several centimeters . at this point do not adjust the blade or chuck for the remainder of the experiment .
prepare the razor blades : prepare 2 razor blades per sample . using a metal file , dull the sharped edge of blades .
place mice in a sealed chamber and euthanize mice by isoflurane inhalation and cervical dislocation , then spray 70% ethanol onto the abdomen and thorax .
make a small incision in skin of the abdomen with scissors and then make an incision to expose the peritoneal cavity .
cut the large intestine at the anal verge and tease apart the mesentery until the colon is free .
excise a distal colon segment between 0 and 6 cm from the anus . here , the crypt depth is ~150 m ( see figure 1b )
pin the tissue to the dissection dish with the mucosal surface facing up . stretch the tissue using dissection pins on a silicon dish in hanks buffer and let rest for 10 min at rt ( figure 1c ) .
slide a razor blade under the desired section of tissue and then pour off the hanks buffer .
pick up the razor / tissue sandwich and allow it to warm slightly by placing a finger on the top blade ( mucosal side up .
this orients the tissue so that the basal muscle layers are sectioned first . ) .
separate the sandwich and cut around the edges of the tissue segment . cut a tissue segment approximately , 0.5 cm x 1 cm . edge areas have a tendency to curl , causing the serial sections to contain many tissue layers .
note that over - cutting is better than under - cutting , so err on the side of caution .
allow the tissue to warm until the ice on top of the tissue begins to melt .
at this point quickly and carefully press the tissue into the oct block in the cryostat .
analysis of mrna or protein , place the samples in tubes with 5 crypt - base , 5 transitional , and 5 surface sections per tube . for rna collection
use 5 - 10 g of total rna for cdna generation . for protein purification , add 0.5 ml of cold ripa lysis buffer ( plus protease inhibitors ) per 5 sections .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c . 
 
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
proceeding toward the lumen , the muscularis mucosa is evident adjacent to the crypt - base epithelial cells , transitional cells are in the middle of the tissue , and finally , surface cell populations face the gut lumen . the serial sectioning method described here
maintains an orientation such that the longitudinal and circular muscularis are encountered first ( figure 2b ) , followed by the muscularis mucosa ( figure 2c ) .
note the appearance of non - muscle interstitial cells in the top left of the image .
the subsequent sections contain epithelial cells and interstitial tissue ( lamina propria ) , starting with the crypt - base cells ( figure 2d ) .
note the absence of a central lumen in most of the crypts , which appear darker due to the closely packed nuclei .
the transitional cells appear as tightly packed glands with prominent central lumens ( figure 2e ) .
lastly , surface cell populations have an irregular structure , with areas of the epithelial monolayer viewed en face ( figure 2f ) . serial sections as described above may also be processed for immunofluorescence labeling and confocal microscopy ( as described here ) . figure 2
g shows isolated crypts fixed and permeabilized in 100% methanol and subsequently stained for nuclei ( blue ) and the cell - cell junction protein zonula occludens 1 ( zo-1 , green ) . in the traditional sectioning orientation , crypt and surface cell can be viewed simultaneously ( figure 2 g ) .
this z0 - 1 lining of the lumen can also be seen in serial sectioned samples of transitional cell populations , in the center of the circular glands ( figure 2 g ) .
enhanced zo-1 stain is seen in surface cell populations ( figure 2i and j ) .
several differentiation factors are known to be expressed in a spatiotemporal fashion along the crypt - to  surface axis .
this includes the transcription factor kruppel - like factor 4 ( klf4 ) , which is known to be enriched in surface cell populations . using traditional sectioning methods
, klf4 is found in the nuclei of lumen facing surface cell populations ( figure 2k ) .
we therefore speculated that klf4 mrna would be predominantly expressed in surface cell populations . to test this ,
serial sections were collected and grouped into surface , transitional , and crypt - base samples .
subsequently , rna was harvested using standard trizol extraction , with an additional purification by rneasy column and dnase treatment .
rna was collected from 5 pooled consecutive sections , which yielded between 5 - 10 g of total rna per sample .
these samples were then subjected to semi - quantitative real time pcr for both klf4 and a reference gene , tata box binding protein 1 ( tbp-1 ) ( figure 2l ) . as shown in figure 2l , after normalization to tbp-1 , surface cells were found to express up to 8 times the klf4 mrna that is expressed in crypt cells .
similarly , klf4 protein levels were found to exhibit spatial regulation along the crypt - surface axis .
serial sections were pooled as described above and then incubated in lysis buffer for 20 min at 4 c .
samples were then subjected analysis by sds - page ( figure 2 m ) . as shown in figure 2 m ,
the above findings demonstrate the ability of this protocol to isolate large amounts of surface and crypt epithelial cells from the murine colonic mucosa .
figure 1 : ( a ) schematic showing murine colonic mucosa and external muscle layers .
our method aims to collect discrete surface cell and crypt - base cell populations by serial cryosectioning ( 10 m each ) .
( b ) crypt height varies along the length of the colon ( distance between crypt - base and surface layers ) .
data points indicate individual crypt measurements and symbols indicate biological replicates ( n = 4 ) .
( c ) colon segments collected , bisected , and pinned onto a silicon dissecting dish .
( d ) a tissue segment approximately 3 cm from the anus is pressed between to razor blades and the frozen on dry ice .
( a ) colonic tissue stained with hematoxylin - eosin in cross - section showing the circular muscularis ( c.m . ) , the muscularis mucosa ( m.m . ) , crypt - base cells , transitional cells , and surface cells .
( d ) crypt - base cells . note the absence of a central lumen .
( f ) surface cells . ( g ) immunofluorescence staining of isolated colonic crypts .
zo-1 ( green ) and nuclei ( blue ) are observed in cross - section .
( l ) real - time pcr assessment of klf4 mrna levels between the three compartments .
this includes gaps in our understanding of the cellular signaling environment of the stem cell compartment at the base of the colonic epithelial crypts .
for example , increased understanding of in vivo differentiation is required as a benchmark for studies aimed at producing in vitro primary intestinal systems .
firstly , removing the edges around the frozen tissue segment ( as discussed in section 3.2 ) is required as the tissue edges curl during dissection and freezing .
failure to move these edges results is a mixed population of surface and crypt base cells .
mounting the tissue on a pre - chilled , leveled oct block is also essential .
this protocol can be adapted to other areas of the distal colon by altering the number of sections in each pool .
for example , as shown in figure 1b , mucosal crypt length varies between 150 - 300 m .
therefore , when studying the region between 4 cm-6 cm from the anus , the number of sections should be increased from 15 to 30 .
importantly , this protocol is not suggested for the proximal colon ( 7 cm-9 cm ) due to folds ( plicae obliquae ) which extend into the lumen making it impossible to separate surface and crypt - base cells by serial sectioning .
serial sectioning techniques have been used to study crypt - base and surface epithelial cell populations in humans . however
, our protocol adds additional steps that are required due to the small size of murine tissue .
we propose that the above protocol will allow for the investigation of crypt - base and surface epithelial cell population in genetically tractable mouse systems . indeed ,
however , it should be noted that , like several of the alternative methods described above , the resulting sections contain a mixture of epithelial and interstitial lamina propria cells . in conclusion ,
the protocol described here provides a rapid , high yield method for the analysis of molecular processes in spatially distinct regions of the murine colonic mucosa .
 the colonic mucosal tissue provides a vital barrier to luminal antigens . this barrier is composed of a monolayer of simple columnar epithelial cells . 
 the colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of lieberkhn . 
 progenitor cells produced in the crypt - bases migrate toward the luminal surface , undergoing a process of cellular differentiation before being shed into the gut lumen . in order to study these processes at the molecular level , 
 we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa ; the proliferative crypt zone , and the differentiated surface epithelial cells . 
 our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of rna and protein . 
Introduction
Protocol
1. Experimental Setup
2. Dissection of Distal Colonic Tissue
3. Mount Tissue on the Cryostat for Sectioning
Representative Results
Discussion
Disclosures