TITLE: High resolution labeling of mucosal vagal afferent fibers using Dextran-Biotin with counterstaining
AUTHORS: Terry  Powley, Jennifer  Mcadams, Robert  Phillips, Deborah  Jaffey

[DESCRIPTION]
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.

[STEPS]
SECTION: Animals
1. Two- to four-month-old male
 
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
 
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.
SECTION: Neural tracer injections
2. Overnight-fasted animals were anesthetized with 
 
starting at an initial concentration of 5%, dropping to 2% or less as required to maintain anesthesia.  After anesthesia, 
 
(0.2 mg/ml, s.c.) was injected to minimize secretions.
SECTION: Neural tracer injections
3. 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 
 
and 
 
Injections were performed with a 
 
and
 
To control the injection placement and to check the distribution of the tracer within the ganglia under direct visual control, 
 
was added to the dextran tracer (0.01 mg per 100 μl solution).
SECTION: Neural tracer injections
4. 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. 
 
(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.
SECTION: Perfusion, GI Dissection and Fixation
5. 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
 
and 
 
(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.
 
(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.


SECTION: Perfusion, GI Dissection and Fixation
6. 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.


SECTION: DAB labeling
13. Rinse 3x10min in 0.3% PBST, and the incubate for 48 hours in avidin– biotin–horseradish complex 
 
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using), at 4°C
SECTION: Neurite Tracing and Morphometry
26. 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
 
(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.
SECTION: Gelatin Embedding
7. 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)
 
Remove from the heat and add the gelatin and stir until dissolved.
 
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.
SECTION: Gelatin Embedding
8. 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
 
Time the program so that it ends at an appropriate time the following morning.
SECTION: DAB labeling
12. 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.
SECTION: DAB labeling
14. Rinse in PBS (3x10min) and then react with DAB
 
( 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.
SECTION: Slide mounting
25. 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
 

SECTION: Gastrin Counterstaining
15. 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.
 

 

SECTION: Gastrin Counterstaining
16. Rinse 3x10min in PBS, followed by 2 hours in Avidin blocking solution
 
Rinse 3x10min in PBS followed by 2 hours in Biotin blocking solution
 
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)
 

SECTION: Ghrelin Counterstaining
20. Rinse 3x10min in PBS, then soak for 48 hours in 2% PBST, 0.08% Na Azide, 5% NGS, 2% BSA at 4°C.
 

 

SECTION: Gastrin Counterstaining
17. 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)
 

SECTION: Gastrin Counterstaining
18. Rinse 3x10min in 0.3% PBST, followed by 48 hrs @ 4°C in 
 
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using)
SECTION: Gastrin Counterstaining
19. Rinse 3x10min in PBS followed by 20 min in
 
Rinse 3x10min in cold dH2O
SECTION: Ghrelin Counterstaining
21. Rinse 3x10min in PBS, followed by 2 hours in Avidin blocking solution
 
Rinse 3x10min in PBS followed by 2 hours in Biotin blocking solution
 
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)
 

SECTION: Ghrelin Counterstaining
22. 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)
 

SECTION: Ghrelin Counterstaining
23. Rinse 3x10min in 0.3% PBST, followed by 48 hours at 4°C in 
 
(bottle A and bottle B each diluted 1:50 in 0.5% PBST, mixed 30 min before using)
SECTION: Ghrelin Counterstaining
24. Rinse 3x10min in PBS followed by 20 min in
 
Rinse 3x10min in cold dH2O
SECTION: Gelatin Embedding
11. 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.
 

SECTION: Gelatin Embedding
8. 
SECTION: Gelatin Embedding
9. 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.
 

SECTION: Gelatin Embedding
10. 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.