Sep 21, 2022

Public workspacePreparation and Immunohistochemistry of Mouse Small Intestine and Colon

DOI
dx.doi.org/10.17504/protocols.io.j8nlkw575l5r/v1
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkw575l5r/v1
Protocol Citation: Nathan D Rossen 2022. Preparation and Immunohistochemistry of Mouse Small Intestine and Colon. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw575l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 19, 2022
Last Modified: September 21, 2022
Protocol Integer ID: 70249
Abstract
Mice were euthanized, the small and large intestine collected, and fixed frozen sections prepared. The microscope mounted tissue sections were stained using immunohistochemistry. Expression of specific proteins and reporter proteins were visualized using microscopy.
Materials

Primary Antibodies
ABCDEF
TargetHostDilutionManufacturerCatalog numberRRID
SerotoninRabbit1:5000ImmunoStar Cat# 20080 RRID:AB_572263
SerotoninGoat1:500AbcamCat# ab66047RRID:AB_1142794
GFPChicken1:500Aves LabsCat# GFP-1010RRID:AB_2307313
dsRedRabbit1:500Takara BioCat# 632496RRID:AB_10013483
Secondary Antibodies
ABCDEF
Conjugate/TargetHostDilutionManufacturerCatalog numberRRID
AF647 anti-rabbitDonkey1:500Invitrogen Catalog # A32795 RRID:AB_2536183
AF647 anti-goatDonkey1:500Invitrogen Catalog # A-21447 RRID:AB_141844
AF488 anti-chickenDonkey1:500Invitrogen Catalog # A78948 RRID:AB_2921070
AF564 anti-rabbitDonkey1:500Invitrogen Catalog # A10042 RRID:AB_2534017

Other Reagents
ReagentOCT (Optimal Cutting Temperature compound)Sakura FinetekCatalog #4583
ReagentDonkey SerumEmd MilliporeCatalog #S30-100ML
ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML
ReagentBovine Serum AlbuminSigma AldrichCatalog #A7030
ReagentSucroseSigma AldrichCatalog #S7903
ReagentProlong GoldThermo Fisher ScientificCatalog #P36930
ReagentParaformaldehyde 32% (methanol free)Electron Microscopy SciencesCatalog #15714


Composition of Abbreviated Solutions
PBS: phosphate-buffered saline, 0.1 M, pH 7,2
Blocking buffer: 5% donkey serum, 5% BSA, .3% Triton X-100 in PBS
Tissue Collection and Preparation
Tissue Collection and Preparation
Mice 6 - 8 weeks old were euthanized by isolfurane administration followed by cervical dislocation.
Mice were transcardially perfused with 5ml ice cold PBS.
Whole small intestine and colon were collected and kept in PBS on ice. The small intestine was dissected into three segments of approximately equal length.
Each segment (proximal, medial, and distal small intestine and whole colon) was flushed with 5ml ice cold PBS, cut lengthwise, and laid flat with the mucosal surface facing up. Each segment was further washed with ice cold PBS.
Tissue segments were washed with ice cold 4% PFA in PBS, and then laid flat in between two layers of filter paper and fixed in 4% PFA in PBS at 4C for either 4hrs or 24hrs.

Tissue was rinsed in excess PBS and transferred to 30% sucrose solution for at least 16 hours.

Tissue was transferred to a mixture of 50% OCT, 15% sucrose in PBS and rolled on a wooden stick.
Tissue was frozen in OCT as a "swiss roll" and sectioned into 5um or 20um sections on a cryostat and kept at -80C.
Immunohistochemistry
Immunohistochemistry
Slide-mounted tissue sections were thawed and air dried for 30 minutes
Sections were washed twice for 10 minutes in PBS and once for 10 minutes in .2% Triton X-100 in PBS.
Sections were blocked for 1 hour in blocking buffer at room temperature.
Sections were incubated with primary antibodies in blocking buffer overnight at 4C.
Sections were washed thrice for 10 minutes in .2% Triton X-100 in PBS.
Sections were incubated with secondary antibodies and DAPI in blocking buffer for 1-2 hours at room temperature.
Sections were washed twice for 10 minutes in .2% Triton X-100 in PBS and twice for 10 minutes in PBS.
Sections were mounted in Prolong Gold Mounting Media.