euthanized mice by local iucac guidelines pin each limb on styrofoam surface and rinse abdominal surface with 70% ethanol . make one cut at the distal esophagus and a second cut at the distal large intestine and remove the gi tract from stomach to anus en bloc , carefully cutting ligaments at the duodenum and cecum using small scissors . to separate stomach , small intestine , and large intestine , cut at pylorus and ileocecal junction . rinse the lumen of each part of the gi tract with pbs using a 5ml syringe attached to a feeding needle ( older mice ) or blunt needle ( younger mice ) . cut two pieces for each part of the gi tract , one for whole mount visualization and one for cryosection . place the entire piece of each part of gi tract into a 1.5 cm microfuge tube filled with 1.0 ml of ice cold acetone and fix samples for at least 1 hour at 4c . carefully scrape off mucosal layer with a scalpel by holding one end with a pair of tweezers , leaving the muscularis . block and incubation steps can be done in one well of a 24-well plate or in a 1.5ml microfuge tube . block with 0.5 ml of blocking buffer ( 5% goat serum , 0.1% triton x-100 in pbs for 1 hour at 4 c ) . we have successfully used ack-2 rat anti - kit , rabbit anti - ano1 , and rabbit anti - pgp9.5 antibodies for whole mount immunostaining . incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature on rotator . we typically use alexa fluor 488 anti - rabbit and alexa fluor 594 anti - rat antibodies . mount on slide , serosal side on top to ensure the orientation of tissue during microscopy . this orientation will ensure the serosal side to face the coverslip and be encountered first as one focuses into the slide on a microscope . sometimes , we briefly examine the tissue under fluorescence microscopy to properly orient . under fluorescence microscopy to visualize kit or ano1 , one should encounter the icc - my network first from the side of the coverslip . add 50 l of hard set mounting media ( we use prolong gold ) and gently place cover slip on top of tissue . allow setting of mounting overnight at room temperature and store slides at -80 c . for microscopic examination , we used a wide - field microscope with a z - drive . we used either a 20x air objective ( na 0.75 , plan apochromat ) or 60x oil objective ( na 1.4 , plan apochromat ) and took 2 m or 1 m z - sections that span the entire muscularis . place each part of the gi tract , serosal side down , flat on filter paper . place tissue on filter paper in 4% paraformaldehyde ( pfa ) in pbs ( we dilute 32% paraformaldehyde in sealed vial into pbs right before use ) for 2 hours at room temperature . fixation can be done in a 6- or 12-well plate depending on size of tissue , or in a 1.5 ml microfuge tube . if done in plate , parafilm to minimize pfa exposure . longer fixation times may result in antigen masking , particularly for the ack-2 anti - kit antibody . when examining mice with different genotypes or with different treatments , it may be useful to mount tissue from different mice onto same cryomold to ensure parallel processing . freeze on dry ice and place in -80 c freezer for storage . cut 10 m cryosections onto slide and store at -80 c freezer . block with 150 l of blocking buffer ( same as for whole mount immunostaining ) for 1 hour . aspirate blocking buffer and add 150 l primary antibody diluted in blocking buffer and incubate overnight at 4 c in a humidified chamber . we typically use alexa 488 anti - rabbit and alexa 594 anti - rat antibodies . wash for 15 min 3x with pbs . add a drop of hard set mounting media with dapi ( we use prolong gold ) and place # 1.5 coverslip . acquire triple fluorescence images using dapi , fitc , and texas red filter sets . here stain of a sucrose protected fixed frozen section used for immunofluorescence shows some artifacts compared to a reference paraffin section ( figure 2 ) , where the yellow line demarks the myenteric plexus between the circular and longitudinal muscle layers and the green line demarks the border between the mucosa and muscularlis . examining the cryosection , from the serosa to mucosa , you first encounter the thinner longitudinal muscle ( lm ) layer followed by the thicker circular muscle ( cm ) layer . the section should be in parallel with the longitudinal muscle so the nuclei of lm should be somewhat elongated while the nuclei of the cm should be small and round . between the lm and cm the myenteric icc network ( icc - my ) surround the myenteric plexus and are made of multipolar cells . within the longitudinal muscle layer , there are rare intramuscular iccs ( icc - ims ) which are bipolar cells running in parallel with the muscle . within the circular muscle layer , there are abundant icc - ims which are also bipolar cells that run parallel with the circular muscle and are cross - sectioned with this cut . at the junction of the muscularis and submucosa , the submucosal icc network consisting of icc - smps is a flat network of multipolar iccs . examining the whole mount kit immunostaining of the large intestine , one should expect to encounter the icc - my network first , and then the icc - im of the cm , and icc - smp networks , as one focuses from the coverslip ( figure . 3 ) . volumetric measure of icc networks using 3d reconstruction of whole - mount images have been described . using wide - field microscope , there is significant out - of - plane fluorescence in the raw images which can be removed after deconvolution ( figure 3 ) . ano1 is another marker of icc s and co - immunostaining of kit and ano1 shows complete overlap of icc staining ( figure 4 ) . a ) representative h&e staining of the mouse large intestine of cryosections prepared as described and reference paraffin embedded section showing some shrinkage artifacts of cryosections . the yellow line demarks the myenteric plexus and the blue line separates the mucosa from the muscularis . b ) representative kit ( red , ack-2 ) and pgp9.5 ( green ) double immunofluorescence with dapi ( blue ) counterstaining of the mouse large intestine . representative kit ( red , ack-2 ) whole - mount immunofluorescence of the same field at three focal planes , the icc - my plane at the lm / cm border , the icc - im plane in the cm , and the icc - smp plane at the cm / submucosa border . representative double - immunofluorescence of ano1 ( green ) and kit ( red , ack-2 ) in large intestine . euthanized mice by local iucac guidelines pin each limb on styrofoam surface and rinse abdominal surface with 70% ethanol . make one cut at the distal esophagus and a second cut at the distal large intestine and remove the gi tract from stomach to anus en bloc , carefully cutting ligaments at the duodenum and cecum using small scissors . to separate stomach , small intestine , and large intestine , cut at pylorus and ileocecal junction . rinse the lumen of each part of the gi tract with pbs using a 5ml syringe attached to a feeding needle ( older mice ) or blunt needle ( younger mice ) . cut two pieces for each part of the gi tract , one for whole mount visualization and one for cryosection . place the entire piece of each part of gi tract into a 1.5 cm microfuge tube filled with 1.0 ml of ice cold acetone and fix samples for at least 1 hour at 4c . carefully scrape off mucosal layer with a scalpel by holding one end with a pair of tweezers , leaving the muscularis . block and incubation steps can be done in one well of a 24-well plate or in a 1.5ml microfuge tube . block with 0.5 ml of blocking buffer ( 5% goat serum , 0.1% triton x-100 in pbs for 1 hour at 4 c ) . we have successfully used ack-2 rat anti - kit , rabbit anti - ano1 , and rabbit anti - pgp9.5 antibodies for whole mount immunostaining . incubate with secondary antibody diluted in blocking buffer for 2 hours at room temperature on rotator . we typically use alexa fluor 488 anti - rabbit and alexa fluor 594 anti - rat antibodies . mount on slide , serosal side on top to ensure the orientation of tissue during microscopy . this orientation will ensure the serosal side to face the coverslip and be encountered first as one focuses into the slide on a microscope . sometimes , we briefly examine the tissue under fluorescence microscopy to properly orient . under fluorescence microscopy to visualize kit or ano1 , one should encounter the icc - my network first from the side of the coverslip . add 50 l of hard set mounting media ( we use prolong gold ) and gently place cover slip on top of tissue . for microscopic examination , we used a wide - field microscope with a z - drive . we used either a 20x air objective ( na 0.75 , plan apochromat ) or 60x oil objective ( na 1.4 , plan apochromat ) and took 2 m or 1 m z - sections that span the entire muscularis . place each part of the gi tract , serosal side down , flat on filter paper . cut filter paper around the tissue . place tissue on filter paper in 4% paraformaldehyde ( pfa ) in pbs ( we dilute 32% paraformaldehyde in sealed vial into pbs right before use ) for 2 hours at room temperature . fixation can be done in a 6- or 12-well plate depending on size of tissue , or in a 1.5 ml microfuge tube . if done in plate , parafilm to minimize pfa exposure . longer fixation times may result in antigen masking , particularly for the ack-2 anti - kit antibody . remove tissue from pfa and place in 30% sucrose in pbs . allow tissue to sink when examining mice with different genotypes or with different treatments , it may be useful to mount tissue from different mice onto same cryomold to ensure parallel processing . freeze on dry ice and place in -80 c freezer for storage . cut 10 m cryosections onto slide and store at -80 c freezer . first equilibrate slide at room temperature for 15 minutes . then draw circle around tissue using pap pen . block with 150 l of blocking buffer ( same as for whole mount immunostaining ) for 1 hour . aspirate blocking buffer and add 150 l primary antibody diluted in blocking buffer and incubate overnight at 4 c in a humidified chamber . we typically use alexa 488 anti - rabbit and alexa 594 anti - rat antibodies . add a drop of hard set mounting media with dapi ( we use prolong gold ) and place # 1.5 coverslip . place slide at room temperature overnight to set and store in -80 c freezer . h&e stain of a sucrose protected fixed frozen section used for immunofluorescence shows some artifacts compared to a reference paraffin section ( figure 2 ) , where the yellow line demarks the myenteric plexus between the circular and longitudinal muscle layers and the green line demarks the border between the mucosa and muscularlis . examining the cryosection , from the serosa to mucosa , you first encounter the thinner longitudinal muscle ( lm ) layer followed by the thicker circular muscle ( cm ) layer . the section should be in parallel with the longitudinal muscle so the nuclei of lm should be somewhat elongated while the nuclei of the cm should be small and round . between the lm and cm are myenteric plexi made of neurons that stain with pgp9.5 . the myenteric icc network ( icc - my ) surround the myenteric plexus and are made of multipolar cells . within the longitudinal muscle layer , there are rare intramuscular iccs ( icc - ims ) which are bipolar cells running in parallel with the muscle . within the circular muscle layer , there are abundant icc - ims which are also bipolar cells that run parallel with the circular muscle and are cross - sectioned with this cut . at the junction of the muscularis and submucosa , the submucosal icc network consisting of icc - smps is a flat network of multipolar iccs . examining the whole mount kit immunostaining of the large intestine , one should expect to encounter the icc - my network first , and then the icc - im of the cm , and icc - smp networks , as one focuses from the coverslip ( figure . 3 ) . volumetric measure of icc networks using 3d reconstruction of whole - mount images have been described . using wide - field microscope , there is significant out - of - plane fluorescence in the raw images which can be removed after deconvolution ( figure 3 ) . ano1 is another marker of icc s and co - immunostaining of kit and ano1 shows complete overlap of icc staining ( figure 4 ) . a ) representative h&e staining of the mouse large intestine of cryosections prepared as described and reference paraffin embedded section showing some shrinkage artifacts of cryosections . the yellow line demarks the myenteric plexus and the blue line separates the mucosa from the muscularis . b ) representative kit ( red , ack-2 ) and pgp9.5 ( green ) double immunofluorescence with dapi ( blue ) counterstaining of the mouse large intestine . ack-2 ) whole - mount immunofluorescence of the same field at three focal planes , the icc - my plane at the lm / cm border , the icc - im plane in the cm , and the icc - smp plane at the cm / submucosa border . representative double - immunofluorescence of ano1 ( green ) and kit ( red , ack-2 ) in large intestine . interstitial cells of cajal were initially characterized by santiago ramn y cajal exactly one century ago using methylene blue and silver chromate stains of gastrointestinal muscularis . cajal initially thought icc s were neurons based on their processes that are reminiscent of axons and dendrites . over many ensuing years , study of icc biology has been limited by lack of specific markers until the discovery of that kit not only is expressed in icc , but is also required for their development . since then , kit immunofluorescence has been widely used in the study of icc biology and has also led to the appreciation of icc in other contractile organs such as the urinary bladder . there have been numerous publications over the past 15 years using immunofluorescence to identify icc using various fixation and mounting techniques . in our hands " fixed - frozen " cryosections that have been prefixed with paraformaldehyde and sucrose protected works best compared to acetone or paraformaldehyde post - fixation after cryosectioning . for whole mounts , we have found that acetone fixation followed by mucosal dissection gives the most robust results . historically , the ack-2 has been the kit antibody of choice for mouse icc identification . it is a rat monoclonal that recognizes the extracellular domain and it is also a blocking antibody that causes ileus when given to live mice . however , the ack-2 epitope is slowly destroyed by paraformaldehyde fixation and is not rescued by standard antigen retrieval methods . compared to fixed frozen blocks and sections , paraffin embedded blocks and sections preserves morphology better and is more amenable to long term archiving ( figure 4 ) . we have characterized that d13a2 , a new rabbit monoclonal antibody , works exceptionally well in both fixed - frozen and paraffin sections of the gi tract . kit is expressed in several other cell types , including melanocytes , hematopoietic cells , germ cells , and in particular mast cells which are also found in the gi tract . luckily , most mast cells are found is the mucosa and not in the muscularis . double staining with rabbit anti - ano1 and rat anti - kit can eliminate non - specific staining of each antibody ( figure 4 ) . it is also worth noting that the relative expression of kit and ano1 between subclasses of icc seems different . for example , in the small intestine , kit staining is much more intense in myenteric icc compared to icc of the deep muscle plexus whereas ano1 staining is comparable .
the interstitial cells of cajal ( icc ) are mesenchymal derived " pacemaker cells " of the gastrointestinal ( gi ) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1 . 
 different subtypes of icc form distinct networks in the muscularis of the gi tract 2,3 . loss or injury to these networks 
 is associated with a number of motility disorders4 . 
 icc cells express the kit receptor tyrosine kinase on the plasma membrane and kit immunostaining has been used for the past 15 years to label the icc network5,6 . 
 importantly , normal kit activity is required for icc development5,6 . 
 neoplastic transformation of icc cells results in gastrointestinal stromal tumor ( gist ) , that frequently harbor gain - of - function kit mutations7,8 . 
 we recently showed that etv1 is a lineage - specific survival factor expressed in the icc / gist lineage and is a master transcriptional regulator required for both normal icc network formation and for of gist tumorigenesis9 . 
 we further demonstrate that it cooperates with activating kit mutations in tumorigenesis . here 
 , we describe methods for visualization of icc networks in mice , largely based on previously published protocols10,11 . 
 more recently , the chloride channel anoctamin 1 ( ano1 ) has also been characterized as a specific membrane marker of icc11,12 . because of their plasma membrane localization 
 , immunofluorescence of both proteins can be used to visualize the icc networks . here , we describe visualization of the icc networks by fixed - frozen cyrosections and whole mount preparations .