Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

1. Animals Breeding and Trophic Peptide Treatment

1. Breed C57BL/mice by placing the female mice (~6 week old, average weight 20 g) into male home cages for 2 hr.
2. Check for a sperm plug and/or vaginal smear immediately afterwards.
3. If positive, designate this time point as embryonic day 0 (E0).
4. On E7, weight-match pregnant females to control and treatment groups (3 groups) are assigned as described recently ⁴: 1) Alcohol (ethanol) liquid diet group (ALC); 2) pair-fed control group (PF); 3) Peptide treatment group, which should receive intraperitoneal (i.p.) injection of ADNF-9 peptide alongside alcohol exposure liquid diet (ALC/ADNF-9). The details of the preparation of ALC and PF diets and the procedure of i.p. injections can be found in our recent work ⁴.
5. Measure daily alcohol liquid diet that can be provided as free access as the sole source of nutrients. PF liquid diet yoked individually to an ALC dam can be measured daily as well.
6. The volume of liquid diet consumed during the previous 24 hr can be recorded from 30-ml graduated screw-cap tubes and freshly prepared diet should be provided daily from E7 to E13.

2. Dissection of Fetuses and Microdissection of Fetal Brains

1. Sacrifice pregnant mice on E13 by deeply euthanizing the mice with a CO₂ procedure followed by a cervical dislocation.
2. Clean the abdominal site of incision with 70% ethanol.
3. Make an abdominal incision using sterile scissors and forceps.
4. Once the abdomen is open, dissect out the uterus by holding it with one forceps and using the other forceps to tear the mesometrium away from the uterus. Make sure to do this procedure slowly in order to avoid puncturing the embryos.
5. Remove the embryos from the uterus and transfer them into a cell culture dish (60 mm x 15 mm) containing Hanks’ Balanced Salt solution (HBSS) in ice cold water.
6. Dissect fetal brains from the whole embryos under stereomicroscope for microdissection.
7. Using ultra-fine forceps for microdissection, pull off the skin of the embryos at the top part of the head to expose the skull.
8. Once the skull is exposed and clearly visualized under the microdissection stereomicroscope, then peel off the skull starting from the posterior part of the brain at the spinal cord and brainstem areas.
9. Be sure to peel off the skull piece by piece from posterior to anterior parts of the head.
10. Once fetal brains are visually exposed, proceed with extracting them.
11. Dissect the fetal brains from the base of the primordium olfactory bulb to the base of the metencephalon.
12. Postfix all dissected fetal brains in 4% paraformaldehyde for 2-3 days. You can also freeze other fetal brains from each dam if you are testing them for Western blot or enzymatic assays.

Note: The procedures for animal uses were approved by the Institutional Animal Care and Use Committee of University of Toledo which are in accordance with the guidelines of the Institutional Animal Care and Use Committee at the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.

3. Embedding Fetal Brains in Gelatin for Sectioning

1. Prepare 10% of gelatin grade A.
2. Fill a peel-away embedding mold halfway and wait until the gelatin solidifies.
3. Place control and prenatally treated fetal brains side-by-side on top of the solidified gelatin. This is to keep consistent conditions of immunodetection of cell death between both the control and experimental groups.
4. Add liquid gelatin on top of the fetal brains to make a complete mold (make sure the gelatin is not too hot).
5. Refrigerate the prepared gelatin mold that holds fetal brains for 15 min.
6. Peel off the embedding mold plastic and then transfer the pre-made gelatin containing fetal brains into 4% paraformaldehyde for two days.
7. Place and section fixed fetal brains in gelatin using vibratome sectioning machine (Leica VT 1000S) at 50 μm thickness for coronal sectioning. This thickness is tested in our studies because it provides better anatomical features. In addition, thicknesses of 25 μm and higher can also be tested with this technique involving embedded of fetal brains in 10% gelatin. Note that coronal sections were cut using vibratome at the basal ganglia eminence level on the forebrain
8. Collect the fetal brain sections in phosphate-buffered saline (PBS) solution.
9. Mount the fetal brain sections in Superfrost Plus slides in water.
10. Dry the mounted sections for 15 min.
11. Place the slides (mounted with the fetal brain sections) in PBS for next day testing of TUNEL reaction.

4. TUNEL Reaction for Detection of Cell Death or Apoptosis

(TUNEL: TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase). This assay is aimed to determine cell death.

1. Treat fetal brain sections mounted in Superfrost Plus slides with Proteinase K (20 μg/ml) for 5 min at 37 °C (300 μl of proteinase K mixed in 20 ml PBS). Slides are assembled in 5-Plend open maile).
2. Rinse fetal brain sections (mounted in slides) with PBS three times for 5 min under Orbital shaker.
3. Incubate the sections with 3% H₂O₂ in methanol for 10 min at room temperature (3 ml 30% H₂O₂ in 27 ml 100% methanol) (slides should not be in a shaker).
4. Rinse sections with PBS three times for 5 min in orbital shaker.
5. Incubate sections in permeabilization solution (0.1% TritonX-100 in 0.1% sodium citrate) for 2 min on ice (4 °C) (100 μl TritonX + 0.1 g sodium citrate + 99.9 ml distilled water).
6. Rinse sections in PBS two times for 5 min in orbital shaker.
7. Dry area around sections in the slides.
8. Draw barrier on Superfrost Plus slides using PAP Pen. This is aimed to prevent any leaking of solution that is used to treat the sections for the detection of TUNEL-positive cells.
9. Incubate the sections with TUNEL reaction mixture (50 μl from bottle 1 and 450 μl from bottle 2) for 1 hr at 37 °C, the control will be used by incubation in solution from bottle 2.
10. Rinse with PBS three times 5 min under Orbit shaker.
11. Dry area around tissue.
12. Incubate sections with converter-POD for 30 min at 37 °C.
13. Rinse the sections three times for 5 min with Tris HCl (pH 7.5, 0.05 M).
14. Incubate the sections for 7 min in 0.05% 3′-3′-diaminobenzidine tetrahydrochloride (DAB) and 0.003% H₂O₂ (25 ml Tris HCl, pH7.5, 0.05 M + 25 μl 3% H₂0₂ + 500 μl DAB) under Oribtal shaker at 4 °C (in ice) and in dark (make sure to do this procedure in new 5-Plend open mailer).
15. Rinse the sections with PBS three times 5 min under Orbital shaker.
16. Keep the slides sections in PBS for no more than 24 hr in PBS, then process them for Nissl staining as detailed in the next section protocol.

Note: DAB is a carcinogen. Use gloves and clean all dishes used with the bleach once finished with all the experiments.

5. Nissl staining for Preparation of Fetal Brain Sections for Microscopic Observation

1. Nissl staining procedure should be done in a biological safety hood.
2. Rinse the slide sections with deionized water for 2 min.
3. Incubate the sections in 70% ethanol for 2 min.
4. Incubate the sections in 95% ethanol for 6 min.
5. Incubate the sections in 70% ethanol for 2 min.
6. Rinse the sections in deionized water for 2 min.
7. Incubate the sections in Cresyl Violet Stain for 3 min.
8. Rinse the sections in deionized water for 2 min.
9. Incubate the sections in 70% ethanol for 2 min.
10. Incubate the sections in 95% ethanol + 3 ml glacial acetic acid for 3 min.
11. Incubate the sections in 95% ethanol for 2 min.
12. Incubate the sections in 100% ethanol for 2 min three times.
13. Incubate the sections in Xylene for 10 min three times.
14. Slides holding the fetal brain sections can be mounted using Permount and cover slip glass slides.
15. Leave the mounted slides overnight prior to microscopic observation and analyses.
16. Microscopic observation and photomicrographs can be performed under Leica upright microscope.