Materials and reagents

Biological materials

1. Fertilized White Leghorn chicken, provided by the Central Avian Research Institute in Bareilly, Uttar Pradesh, India, and reared in our on-campus poultry farm

1. γ-H2AX, 1:500 (Fine Test, catalog number: FNab10441)

2. HIF-1α, 1:1,000 (Novus Biological, catalog number: NB100-449)

3. NOX2, 1:500 (Fine Test, catalog number: FNab05804)

4. IFN-γ, 1:1,000 (Abcam, catalog number: ab133566)

5. RIPK1, 1:500 (Cloud-Clone Corp., catalog number: MAE640Hu21)

6. Cleaved caspase-3, 1:500 (ImmunoTag, catalog number: ITT07022)

7. LC3 l/ll, 1:1,000 (Cell Signaling Technology, catalog number: 4108S)

8. GAPDH, 1:1,000 (Cloud-Clone Corp.,catalog number: MAB932Hu21)

9. Anti-mouse IgG, 1:3,000 (Cell Signaling Technology, catalog number: 7076S)

10. Anti-rabbit IgG, 1:10,000 (Jackson Immuno Research Laboratories, catalog number: 711-035-152)

11. ELISA Kit, TNF-α (Fine Test, catalog number: EH0302)

12. ELISA Kit, IL-1β (Fine Test, catalog number: ECH0040)

13. SDS-PAGE (Invitrogen, catalog number: A25977)

14. Endothelin-1 (ET-1), 1 pM (Sigma-Aldrich, catalog number: E7764)

15. N-Acetyl-L-Cysteine (NAC), 100 µM (Sigma-Aldrich, catalog number: A7250)

1. 70% ethanol (see Recipes)

2. 1× phosphate buffer saline (PBS) (see Recipes)

3. Ringer’s solution (see Recipes)

4. ET-1 (see Recipes)

5. N-Acetyl-L-Cysteine (NAC) solution (see Recipes)

1. 70% ethanol (100 mL)

   Combine 30 mL of sterile water with 70 mL of 200 proof/LR grade ethanol to make a final volume of 100 mL.

   Reagent	Final concentration	Amount
   Ethanol	n/a	70 mL
   Distilled Water	n/a	30 mL
   Total	n/a	100 mL




2. 1× phosphate buffer saline (PBS) (100 mL)

   To make 100 mL of 1× PBS, combine all reagents with 70 mL of distilled water. Completely dissolve the reagents and adjust to the final volume. Autoclave the solution for 15 min at 121 °C. If needed, adjust the pH to 7.4 by adding a few drops of 0.1 N HCl or 0.1 N NaOH. Divide the solution into 10 mL aliquots and store them at room temperature (RT) in sterile 15 mL centrifuge tubes. Store at RT for up to 12 months.

   Reagent	Final concentration	Amount
   Na2HPO4·7H2O	5.37 mM	0.144 g
   NaCl	136.8 mM	0.8 g
   KCl	26.8 mM	0.2 g
   KH2PO4	14.6 mM	0.2 g
   H2O	n/a	100 mL
   Total	n/a	100 mL




3. Ringer’s solution (100 mL)

   Mix NaCl, CaCl₂, and KCl in 70 mL of sterile distilled water. Adjust the pH to 7.4. Let it dissolve completely and autoclave. Then, filter through a 0.22 µm filter and aliquot into single-use amounts (approximately 10 mL). Store at 4 °C for up to 1–2 months.

   Reagent	Final concentration	Amount
   NaCl	123 mM	0.72 g
   CaCl2	1.53 mM	0.017 g
   KCl	4.96 mM	0.037 g
   H2O	n/a	100 mL
   pH	n/a	7.4
   Total	n/a	100 mL




4. ET-1

   To make a 1 mM stock solution, dissolve 2.49 mg of ET-1 in 1 mL of Milli-Q water. Take 1 µL of the 1 mM ET-1 solution and mix it with 999 µL of Milli-Q water to make a 1 µM concentration solution. To achieve a 1 nM concentration, transfer 1 µL of the 1 µM solution into 999 µL of Milli-Q water. Lastly, dissolve 1 µL of the 1 nM solution in 999 µL of Milli-Q water to achieve a final concentration of 1 pM.

   Reagent	Final concentration	Amount
   ET-1	1 mM	2.49 mg
   Milli-Q	n/a	1 mL
   Total	1 mM	1 mL




5. N-Acetyl-L-Cysteine (NAC) solution

   Dissolve 1.63 mg of NAC in 1 mL of Ringer's solution to make a 10 mM stock concentration. Titrate the stock solution to 1 mM by adding 100 µL of the 10 mM NAC solution to 900 µL of Ringer's solution. Further dilute the resulting 1 mM concentration to 100 µM by combining 100 µL of the 1 mM NAC solution with 900 µL of Ringer's solution.

   Reagent	Final concentration	Amount
   NAC	10 mM	1.63 mg
   Ringer's solution	n/a	1 mL
   Total	n/a	1 mL

Laboratory supplies

1. Egg incubator (Gentek, catalog number: GL-100)

2. Pointed sharp curved scissors (Stoelting, catalog number: 52132-11)

3. Hamilton glass syringe (Hamilton, catalog number: 80087)

4. Needle 26G–26 × 1/2 (0.45 mm × 13 mm) (Dispovan, catalog number: 30722D

5. Needle 18G–18G × 1.5 (1.25 mm × 38 mm) (Romsons, catalog number: 13990)

6. Syringe (5 mL) (Dispovan, catalog number: 653054NE1)

7. Tube stand (1.5 mL) (TARSONS)

8. Ocular forceps (Stoelting, catalog number: 52100-52)

9. Ocular iris (Stoelting, catalog number: 52130-00)

10. Packaging tape (Sunrise)

11. Autoclaved tissue roll (Texam Technologies Pvt. Ltd.)

12. Overhead projection pens (OHP) Marker (Camlin)

13. Autoclaved bag (TARSONS, catalog number: 550022)

14. Petri dish (TARSONS, catalog number: 460050)

15. Egg rack

16. Sharp waste container (TARSONS, catalog number: 882210)

17. Centrifuge tube (1.5 mL) (TARSONS, catalog number: 500010X)

18. Beaker (100 mL) (GLASSCO, catalog number: 23.205.03)

19. Pasteur pipette (3 mL) (TARSONS, catalog number: 940050)

Equipment

1. New standard^TM stereotaxic rat and mouse surgery apparatus (Stoelting, catalog number: 51500)

2. Laser Doppler blood flow meter (Moor instruments, catalog number: moorVMS-LDF1)

3. Stereo zoom microscope (Olympus, catalog number: SZ2-STU3)

Software and datasets

1. SPSS Statistics Software IBM 29.0

2. moorVMS-LDF1

3. GraphPad Prism 5 Software

Procedure