Cut the small intestines out of animals .
{"action": "cut", "output": ""}
remove the mesentery #####%%%%% output "PBS" are used in line6
{"action": "remove", "output": "PBS", "container": ["the mesentery"], "reagent": [""], "device": [""]}
After gently pushing out the fecal content using a forceps, wash the small intestines gently twice in PBS .
{"action": "rinse", "output": "", "reagent": ["PBS"]}
cut open longitudinally using a scissor
{"action": "cut", "output": "", "device": ["a scissor"]}
Cut intestinal tissue sections at 5 μm using a Leica CM3050 cryostat, mount on glass microscope slides, .
{"action": "cut", "output": "", "length": ["5 \u03bcm"], "device": ["Leica CM3050 cryostat"]}
subsequently fix for seven minutes in ice-cold acetone for one hour at room temperature
{"action": "fix", "output": "", "time": ["seven minutes"], "temperature": ["ice-cold acetone"]}
Fixed sections can be stored at -80°C in desiccant bags.
{"action": "store", "output": "", "temperature": ["-80\u00b0C"], "container": ["desiccant bags"]}
Using a Dako Pen, mount hydrophobic barrier rings onto glass slides by circling tissues to limit reagent use for staining. #####%%%%% output "staining" are not used
{"action": "handle", "output": "staining", "device": ["a Dako Pen"], "reagent": [""]}
By doing so approximately 400 μl of liquid \(blocking solutions) per slide is required.

Block unspecific binding of antibodies by incubating tissue sections in TBS-T supplemented with 10% normal rabbit serum, for 30 minutes in a humidified chamber. #####%%%%% output "TBS-T" are used in line22
{"action": "block", "output": "TBS-T", "reagent": ["antibodies", "TBS-T"]}
After removal of the blocking solution, dip the slides briefly in TBS-T . #####%%%%% output "TBS-T" are used in line24
{"action": "dip", "output": "", "reagent": ["TBS-T"]}
then apply mixtures of the fluorochrome labeled antibodies diluted in TBS-T for 45 minutes in the dark in a humidified chamber #####%%%%% output "the staining solutions" are used in line26
{"action": "apply", "output": "the staining solutions", "reagent": ["TBS-T"], "time": ["45 minutes"]}
After removal of the staining solutions, wash the slides three times for ten minutes in TBS-T, once with TBS in slide-beakers applying gentle shaking. #####%%%%% output "DAPI nucleic acid stain" are used in line28
{"action": "rinse", "output": "DAPI nucleic acid stain", "reagent": ["the staining solutions"]}
Stain the slides with DAPI nucleic acid stain for 30 seconds . #####%%%%% output "PBS" are used in line30
{"action": "stain", "output": "PBS", "reagent": ["DAPI nucleic acid stain"], "time": ["30 seconds"]}
wash three times with PBS in slide-beakers applying gentle shaking
{"action": "rinse", "output": "", "reagent": ["PBS"], "device": ["slide-beakers"]}
Acquire images with a Leica DMRA2 fluorescence microscope equipped with a Retiga EXi digital camera using OpenLab software.
{"action": "acquire", "output": "", "device": ["Leica DMRA2 fluorescence microscope"]}
When comparing tissues of diverse genotypes, keep picture acquisition settings for every fluorescent channel constant.
{"action": "keep", "output": ""}
When comparing images of diverse genotypes , adjustments of input was applied for all compared images in an equal manner. #####%%%%% output "images" are used in line40
{"action": "apply", "output": "images", "device": [""]}
Count the numbers of small intestinal LP IgA+ plasma cells in a blinded fashion utilizing ImageJ. #####%%%%% output "small intestinal LP IgA+ plasma cells" are not used
{"action": "estimate", "output": "small intestinal LP IgA+ plasma cells", "device": ["ImageJ"]}
quantify a total of five separate images from sections of six mice per group #####%%%%% output "five separate images" are not used
{"action": "quantify", "output": "five separate images", "reagent": ["images"]}