Jun 07, 2022
High resolution labeling of mucosal vagal afferent fibers using Dextran-Biotin with counterstaining
- 1Purdue University
Protocol Citation: Terry Powley, Jennifer Mcadams, Robert Phillips, Deborah Jaffey 2022. High resolution labeling of mucosal vagal afferent fibers using Dextran-Biotin with counterstaining. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6nx5rgqe/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: January 08, 2020
Last Modified: June 07, 2022
Protocol Integer ID: 31596
Keywords: Mucosal afferent, vagal, tracing, ABC-DAB, gastrin, ghrelin, nodose, rat
Abstract
This protocol describes the methods used to trace and enable morphometric quantification of vagal afferent neurites in mucosal layers of the rat GI system. A mixture of dextran conjugates was injected into the nodose ganglia of young adult Sprague-Dawley rats and after a survival period of 14 days for optimal tracer transport, stomachs and intestines were removed and selected pieces embedded in gelatin, sectioned and processed. ABC-DAB was used to create a permanent gold-brown stain of all labeled afferent neurites. Some samples were also counterstained for ghrelin and gastrin.
Protocol materials
GlycopyrrolateAkorn Inc
Step 2
Normal Donkey SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9663-10ml
Step 15
Bovine Serum AlbuminJackson ImmunoResearch Laboratories, Inc.Catalog #001-000-173
In 2 steps
35-gauge NANOFIL needleWorld Precision InstrumentsCatalog #NF35BV-2
Step 3
3,3-DIAMINOBENZIDINE.4HCl.xH2O Pure 98% * 5 gMerck MilliporeSigma (Sigma-Aldrich)Catalog #32750-5G
Step 14
Gastrin (GAST) Guinea Pig Polyclonal AntibodyOriGeneCatalog #BP5046
Step 16
Biotin Anti-Guinea Pig IgG (H L) made in donkeyJackson ImmunoResearch Laboratories, Inc.Catalog #706-065-148
Step 17
KetaminePatterson VeterinaryCatalog #07-803-6637
Step 5
Goat serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #G9023
Step 20
Ghrelin (Rat, Mouse) - AntibodyPhoenix PharmaceuticalsCatalog #H-031-31
Step 21
2018 Teklad global 18% protein rodent dietEnvigo
Step 1
IsofluraneAkorn IncCatalog #NDC: 59399-106-01
Step 2
Gelatin Type 8 (Bloom strength 100, narrow range)Great Lakes Gelatin
Step 7
Biotin-SP (long spacer) AffiniPure Goat Anti-Rabbit IgG (H L) (min X Hu, Ms, Rat Sr Prot)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-065-144
Step 22
Dextran-Biotin 10k, Lysine fixableThermo Fisher ScientificCatalog #D1956
Step 3
Dextran-Biotin 3k, Lysine fixableThermo Fisher ScientificCatalog #D7135
Step 3
Vectastain Elite ABC HRP kitVector LaboratoriesCatalog #PK-6100
In 3 steps
ThymolFisher ScientificCatalog #AC150331000
Step 7
Avidin/Biotin blocking kitVector LaboratoriesCatalog #SP-2001
In 4 steps
Sprague-DawleyEnvigo
Step 1
HeparinHenry Schein Animal HealthCatalog #049130
Step 5
10 microliter Nanofil syringeWorld Precision InstrumentsCatalog #Nanofil
Step 3
Cytoseal XYLFisher ScientificCatalog #22-050-262
Step 25
VECTOR® SG Peroxidase (HRP) Substrate KitVector LaboratoriesCatalog #SK-4700
In 2 steps
Fast Green FCFMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7252
Step 3
Buprenorphine (Buprenex)Midwest Veterinary SUpplyCatalog # 191.26890.3
Step 4
XylazineAkorn IncCatalog #NDC: 59399-110-20
Step 5
Animals
Animals
Two- to four-month-old male
Sprague-DawleySigma Aldrich
rats in the weight range of 180g to 360g at the time of tracer injection were housed individually in wire hanging cages or in vented rack plastic cages in an Association for Assessment and Accreditation of Laboratory Animal Care-approved temperature (22–24 °C) and humidity (40–60%)-controlled colony room. The room was maintained on a 12-hour light–dark schedule. Pelleted chow
2018 Teklad global 18% protein rodent dietSigma Aldrich
and filtered tap water were provided ad libitum, except for the night before tracer injection, when food but not water was removed. All husbandry practices conformed to the NIH Guide for the Care and Use of Laboratory Animals (8th edition) and were reviewed and approved by the Purdue University Animal Care and Use Committee. All efforts were made to minimize any suffering as well as the number of animals used.
Neural tracer injections
Neural tracer injections
Overnight-fasted animals were anesthetized with
IsofluraneSigma AldrichCatalog #NDC: 59399-106-01
starting at an initial concentration of 5%, dropping to 2% or less as required to maintain anesthesia. After anesthesia,
GlycopyrrolateSigma Aldrich
(0.2 mg/ml, s.c.) was injected to minimize secretions.
For dextran injections, each anesthetized animal was placed in a supine position, and a midline incision of the skin of the ventral neck was made. Both nodose ganglia were then exposed by blunt dissection of the overlying muscles. Each animal received bilateral injections (1 μl per ganglion) of lysine-fixable dextran biotin solution consisting of a 1:1 mixture of 3K and 10K MW dextrans in ultrapure DI water or PBS - final concentration 15% dextran biotin consisting of 7.5% each of
Dextran-Biotin 3k, Lysine fixableSigma AldrichCatalog #D7135
and
Dextran-Biotin 10k, Lysine fixableSigma AldrichCatalog #D1956
Injections were performed with a
35-gauge NANOFIL needleSigma AldrichCatalog #NF35BV-2
and
10 microliter Nanofil syringeSigma AldrichCatalog #Nanofil
To control the injection placement and to check the distribution of the tracer within the ganglia under direct visual control,
Fast Green FCFSigma AldrichCatalog #F7252
was added to the dextran tracer (0.01 mg per 100 μl solution).
After both ganglia had been injected, the muscle and skin incisions were closed with interrupted sutures. The animal was transferred first to a circulating-water heating pad until its righting reflexes had returned and then to its home cage.
Buprenorphine (Buprenex)Sigma AldrichCatalog # 191.26890.3
(0.01 mg/kg) was given s.c. prior to suturing and again the following morning for analgesia. For the evening meal following surgery the animals are given a bowl of "chow mash", chow mixed with water to form a porridge. The next day they are returned to solid food.
Perfusion, GI Dissection and Fixation
Perfusion, GI Dissection and Fixation
After a survival period of 14 days for optimal tracer transport, each rat was given a lethal dose (180 mg/kg−1, i.p.) of sodium pentobarbital or a combination of
KetamineSigma AldrichCatalog #07-803-6637
and
XylazineSigma AldrichCatalog #NDC: 59399-110-20
(275 mg/kg ketamine and 27.5 mg/kg xylazine). The animals had food available ad libitum until they were anesthetized, to facilitate the stomach being full and relaxed in accommodation. When each animal was completely unresponsive to nociceptive pinching and prodding, the GI tract was exposed with a midline abdominal incision.
HeparinSigma AldrichCatalog #049130
(0.5ml, 1000 units/ml) was then injected into the heart and the animal was transcardially perfused through the vasculature, first with physiological saline and then with 4% paraformaldehyde in 0.1 mol liter−1 phosphate-buffered saline (PBS; pH 7.4). Immediately after the beginning of the perfusion, the GI tract was flushed with 4% PF solution to preserve the integrity of the mucosa.
After the perfusion, the distal esophagus and the proximal colon were transected, and the entire GI tract was freed and removed.
4mm blocks of tissue from desired areas of the GI tract were cut out with scissors and placed in 4% paraformaldehyde solution overnight.
Gelatin Embedding
Gelatin Embedding
Prepare 750 ml each of 5%, 10% and 15% gelatin by weight solutions in ultrapure water, as follows:
Add 0.75mg of thymol to 750 ml of ultrapure water and stir on a hot plate at 45°C until dissolved (about 30 min)
ThymolSigma AldrichCatalog #AC150331000
Remove from the heat and add the gelatin and stir until dissolved.
Gelatin Type 8 (Bloom strength 100, narrow range)Sigma Aldrich
Then place the container of gelatin in a water bath at 45°C for one hour with agitation of the bath and occasional stirring of the gelatin.
Place tissue into tissue baskets and into the tissue processor in an initial container of 4% PF, and run a program overnight with the following parameters.
Ultrapure Water @ 25°C - 2hrs
Ultrapure Water @ 25°C - 4hrs
5% Gelatin @ 45°C - 2hrs
10% Gelatin @ 45°C - 2hrs
15% Gelatin @ 45°C - 2hrs
Equipment
Leica TP1020 Semi-enclosed Benchtop Tissue Processor
NAME
Leica Biosystems
BRAND
TP1020
SKU
LINK
Time the program so that it ends at an appropriate time the following morning.
Next morning make another 750 ml of 15% gelatin with 0.1% thymol as above. Fill peel-away molds half-way with 15% gelatin 30min before the end of the tissue processing and allow to harden in the refrigerator for 30 min.
Equipment
Peel Away Disposable Embedding Molds
NAME
Electron Microscopy Sciences
BRAND
70182
SKU
LINK
Fill the molds with more 15% gelatin and position the tissue samples with the desired orientation for sectioning, and allow to harden in the refrigerator for 30min. Carefully cut away mold and place hardened gelatin block in 4% paraformaldehyde solution overnight.
Move gelatin blocks into a water solution 24 hours after placing in 4% paraformaldehyde. Section tissue blocks on a vibratome with a section thickness of 100μm. Place sections into PBS.
Equipment
Leica VT1200 S Fully automated vibrating blade microtome
NAME
Leica Biosystems
BRAND
VT1200S
SKU
LINK
DAB labeling
DAB labeling
Rinse for 3x10min in PBS, followed by a 3% hydrogen peroxide – methanol block (1:4) for 30 min to quench endogenous peroxidase activity.
Then rinse again for 3x10min in PBS, and then soak for 3–5 days in PBS containing 0.5% Triton X-100 and 0.08% Na azide at 4°C to facilitate penetration of reagents.
Rinse 3x10min in 0.3% PBST, and the incubate for 48 hours in avidin– biotin–horseradish complex
Vectastain Elite ABC HRP kitSigma AldrichCatalog #PK-6100
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using), at 4°C
Rinse in PBS (3x10min) and then react with DAB
3,3-DIAMINOBENZIDINE.4HCl.xH2O Pure 98% * 5 gSigma AldrichCatalog #32750-5G
( 0.7 mg/ml DAB, 5.6 ug/ml 3% H2O2 in Tris buffered saline) for 90 minutes at 4°C. Then rinse for 3x10min in cold dH2O.
Gastrin Counterstaining
Gastrin Counterstaining
If no counterstaining is wanted - proceed directly to the slide mounting section. For gastrin counterstaining use this section and then proceed to slide mounting; for ghrelin counterstaining use the next section and then proceed to slide mounting.
Rinse 3x10min in PBS, then soak for 48 hours in 2% PBST, 0.08% Na Azide, 5% NDS, 2% BSA at 4°C.
Normal Donkey SerumSigma AldrichCatalog #D9663-10ml
Bovine Serum AlbuminSigma AldrichCatalog #001-000-173
Rinse 3x10min in PBS, followed by 2 hours in Avidin blocking solution
Avidin/Biotin blocking kitSigma AldrichCatalog #SP-2001
Rinse 3x10min in PBS followed by 2 hours in Biotin blocking solution
Avidin/Biotin blocking kitSigma AldrichCatalog #SP-2001
Rinse 3x10min in PBS, followed by 3 days in primary at 4°C (Gastrin antibody diluted 1:7,500 in 0.5% PBST, 0.08% Na Azide, 5% NDS, 2% BSA)
Gastrin (GAST) Guinea Pig Polyclonal AntibodySigma AldrichCatalog #BP5046
Rinse 3x10min in 0.3% PBST, followed by 48 hours in secondary at 4°C (Biotin Anti-Guinea Pig diluted 1:500 in 0.5% PBST, 0.08% Na Azide, 5% NDS, 2% BSA)
Biotin Anti-Guinea Pig IgG (H L) made in donkeySigma Aldrich
Rinse 3x10min in 0.3% PBST, followed by 48 hrs @ 4°C in
Vectastain Elite ABC HRP kitSigma AldrichCatalog #PK-6100
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using)
Rinse 3x10min in PBS followed by 20 min in
VECTOR® SG Peroxidase (HRP) Substrate KitSigma AldrichCatalog #SK-4700
Rinse 3x10min in cold dH2O
Ghrelin Counterstaining
Ghrelin Counterstaining
Rinse 3x10min in PBS, then soak for 48 hours in 2% PBST, 0.08% Na Azide, 5% NGS, 2% BSA at 4°C.
Goat serumSigma AldrichCatalog #G9023
Bovine Serum AlbuminSigma Aldrich
Rinse 3x10min in PBS, followed by 2 hours in Avidin blocking solution
Avidin/Biotin blocking kitSigma AldrichCatalog #SP-2001
Rinse 3x10min in PBS followed by 2 hours in Biotin blocking solution
Avidin/Biotin blocking kitSigma AldrichCatalog #SP-2001
Rinse 3x10min in PBS, followed by 3 days in primary at 4°C (Ghrelin antibody diluted 1:800 in 0.5% PBST, 0.08% Na Azide, 5% NGS, 2% BSA)
Ghrelin AntibodySigma Aldrich
Rinse 3x10min in 0.3% PBST, followed by 48 hours in secondary at 4°C (Biotin Anti-Rabbit diluted 1:500 in 0.5% PBST, 0.08% Na Azide, 5% NGS, 2% BSA)
Biotin Anti-RabbitSigma Aldrich
Rinse 3x10min in 0.3% PBST, followed by 48 hours at 4°C in
Vectastain Elite ABC HRP kitSigma AldrichCatalog #PK-6100
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using)
Rinse 3x10min in PBS followed by 20 min in
VECTOR® SG Peroxidase (HRP) Substrate KitSigma AldrichCatalog #SK-4700
Rinse 3x10min in cold dH2O
Slide mounting
Slide mounting
Transfer each section to a gelatin coated glass slide, 3-4 sections per slide, maintaining a consistent orientation and section order and label slide with pencil, and allow the slides to dry overnight. Then soak in 4% PF for 1 hour, rinse 3 times with dH2O, and allow the slides to dry overnight. Finally dehydrate 4x4min in ethanol followed by 2x6min xylene, and coverslip with
Cytoseal XYLSigma AldrichCatalog #22-050-262
Neurite Tracing and Morphometry
Neurite Tracing and Morphometry
Scan all sections systematically under a Leica DMRE or DM5500 microscope to identify afferents associated with mucosal layers, villi or crypts/glands and suitable for tracing.
Suitable afferent sections can then be traced using Neurolucida 360 software
Software
Neurolucida
NAME
MBF BioScience
DEVELOPER
(RRID:SCR_001775) controlling the motorized stage of a Zeiss (Oberkochen, Germany) Axio Imager Z2 microscope equipped with DIC optics and long-working-distance (×40 and ×63 oil) objectives. Once tracing of an arbor is complete, morphometric analysis can be performed using the Neurolucida Explorer software.