Song Hong, Ph.D.

Protocols

Protocol for Hematoxylin and Eosin Staining

• The samples are sectioned into 20 um and stored in -20°C until using.
• Slides from each sectioned wound sample with the biggest wound gap are selected
• Immerse slides in acetone and store at -20C for 20 minutes.
• Remove slides from freezer and immerse in 100% ethanol for 2 minutes.
• Immerse in 95% ethanol for 2 minutes.
• Immerse in 80% ethanol for 2 minutes.
• Immerse in DI water for 2 minutes.
• Immerse in Mayer Hematoxylin for 30 seconds.
• Quickly remove from hematoxylin solution and rinse under tap until color no longer streaks across the slide.
• Immerse in DI water for 2 minutes.
• Dip quickly in 0.5% v/v acetic acid solution.
• Immerse in Eosin Y solution for 1 minute.
• Quickly remove from eosin solution and rinse under tap until color no longer streaks across the slide.
• Immerse in 95% ethanol for 2 minutes.
• Immerse in 100% ethanol for 2 minutes.
• Immerse in xylene for 5 minutes.
• Mount in a suitable xylene-based mounting media. Coverslip. Let air dry for 10 minutes.
• Seal the coverslip with clear nail polish. Air dry for 10 minutes.
• Store HE-stained slides at room temperature.
• Analyze using image J.

Protocol for Sirius Red Staining

• Immerse slides in acetone and store at -20C for 20 minutes.
• Make picro-sirius red solution (0.5 g Sirius red in 500 mL saturated picric acid).
• Remove slides from freezer and immerse in 100% ethanol for 2 minutes.
• Immerse in 95% ethanol for 2 minutes.
• Immerse in 80% ethanol for 2 minutes.
• Immerse in DI water for 2 minutes.
• Immerse in hematoxylin for 1 minute.
• Quickly remove from hematoxylin solution and rinse under tap until color no longer streaks across the slide.
• Immerse in DI water for 2 minutes.
• Immerse in picro-sirius red for one hour.
• Rinse under tap.
• Wash in two changes of acidified water (0.5% acetic acid v/v, 5mL in 1 L water).
• Immerse in DI water for 2 minutes.
• Immerse in 95% ethanol for 2 minutes.
• Immerse in 100% ethanol for 2 minutes.
• Immerse in xylene for 5 minutes.
• Mount in a suitable xylene-based mounting media. Coverslip. Let air dry for 10 minutes.
• Seal the coverslip with clear nail polish. Air dry for 10 minutes.
• Store Sirius-stained slides at room temperature in a labeled slide box with experiment info, name, date, etc.

Protocol for MASSON TRICHROME Staining

• Fix slides in 4% formalin – 30 mins
• Hydrate slides in DI water – wash 3X
• Preheat Bouin’s fluid in a water bath to 56-64°C in a fume hood.
• Place slides in the above solution for 60 mins followed by 10 min cooling.
• Rinse in tap water until sections are completely clear.
• Rinse once in DI water.
• Mix equal parts of Weigerts (A) and (B), and stain slides with working Weigert’s Iron Hematoxylin for 5-10 mins. Monitor and add stain as necessary to prevent drying.
• Rinse in running tap water for 2 mins.
• Rinse once in DI water.
• Apply Biebrich Scarlet/ Acid Fuchsin solution for 5-15 mins.
• Rinse in DI water.
• Differentiate in Phosphomolybdic/Phosphotungstic Acid solution for 10-15 mins or until collagen is not red.
• Without rinsing, apply Aniline Blue solution to the slides for 5-10 mins.
• Rinse slides in distilled water.
• Apply acetic acid solution (1%) to the slides for 3-5 mins.
• Dehydrate very quickly in 2 changes of 95% alcohol, followed by 2 chage of absolute alcohol.
• Clear in xylene, mount slides.

Procedures and Lipid Extraction Protocol from Cell Culture (MSC stem cells, control and treatment)

Method

1. When MSC stem cells grow to 80% confluence, cells were used for lipid extraction.
2. MSCs (three 10-cm dishes) were incubates in 50uM DHA in 4ml PBS for 20mins, and then treated with 10ng/ml TNF-alpha, 10ng/ml IL-1 and 100ng/ml LPS for 1hr. Control (three 10-cm dishes) was only incubates with 50uM DHA in PBS without stimulation.
3. After incubation, 1ml ACN was added into solution and stored at -80°C

Lipid Extraction

1. Use 2 volumes of ACN to stop the reaction in the cell culture in a 15-ml-tube for 1 ml culture. Adjust the pH to ~ 4.5-5.0 with ice-cold 0.1M HCl
2. To each sample, add 50ul the internal standard containing 50 ng PGD2-d₄
3. Thaw the culture on ice.
4. Sonicate the culture on ice for 10seconds/pulse x 5.
5. Centrifuge at 5000rpm for 10min at 2°C
6. Transfer the supernatant into a ice-cold 50ml-tube (tube 2)
7. Add ~0.25ml ACN into tube 1 and sonicate cells
8. Centrifuge at 5000rpm for 10min, transfer supernatant into tube 2
9. Repeat 6,7 for twice.
10. Add [pH5.0 NH4Ac-pH5 buffer] in tube 2 to bring the final concentration of ACN to 10%.
11. Wash the SPE cartridge with 1 load of MeOH and then 1 load of deionized water
12. Then pour the extraction in the cartridge and let it go through SPE slowly (dropwise).

Washing

1. When liquid nearly runs out of SPE, turn off SPE, wash each cartridge with 3 loads [NH4Ac-pH5 buffer] to remove proteins.
2. When liquid nearly runs out of SPE, turn off SPE, wash each cartridge with half load of [10%MeOH,NH4Ac-pH5 buffer] to remove yellow staff.
3. Wash each cartridge with 0.5ml deionized-water(pH ~6)
4. 1ml hexane was used to take away water.
5. Let the air go through cartridge for 5min to dry water.
6. Using Kimwipes to absorb the water attached at the tip of the SPE tubes and flow outlet.

Elute analytes out of SPE tubes

1. Turn off the vacuum
2. Put 10 ml-tip-attached-on-500ul-vials under the cartridges to collect ACN/methyl formate fractions.
3. Add 1ml acetonitrile into SPE cartridge to elute lipids.
4. Add 9ml 10% ACN/methyl formate (MF) to each cartridge.
5. Let solvent soak into cartridge for about a minute before opening vacuum
6. Open the vacuum gradually until 10% ACN/MF start to dripping slowly from the outlet of SPE tube
7. Let 10% ACN/MF run through the SPE dropwise (i.e., slowly)

Nitrogen drying

1. Use new glass tips
2. Wash/rinse the 10ml tips with ~1-2ml of MeOH to remove the dust.
3. Place the needle or glass tips ~1cm above the liquid surface of the sample.
4. Dry down solvent gently till ~50 ul liquid left.
5. Using 100 µl ACN/each wash to wash lipids on the wall of 10ml tip down into 500ul-vials
6. Repeat 3 times: 1^st, wash the top of the tip; 2^nd, wash middle of tip; 3^rd wash low end of tip into the 500ul-vials.
7. Make sure no deposit in the solution. If seeing deposits, the solution may need to be filtered with 0.2um nylon syringe-filter.
8. Dry down to final volume of 200ul.
9. Vortex all vials to make sure that the analytes are dissolved and solutions are homogenous. Ready for LC-MS analysis

Protocol for Lipid Extraction from Tissues

1. Sample tissues were cut into small 1-2 mm³ pieces. When cutting the samples, they were located on a plastic cap on ice.
2. The small pieces were transferred to a 5mL tube.
3. 1mL mixture of methanol and NH4Ac-pH4.5 (~10 mM) (MeOH-NH4AC-pH4.5=3:1) was added to each sample.
4. pH was checked and changed to a range of 4.5. pH was regulated by adding 1mL of Acetic acid.
5. 50µL of internal standard (PGD2-d4, NPD1-d2, and DHA-d5) with a concentration of 0.02ng each/µL were added to samples.
6. Then the samples were homogenized using a probe sonicator. The tissues were homogenized for 1 minute [when homogenizing, the tubes were chilled by ice (inside the ice)].
7. After homogenizing samples were centrifuged at 5000rpm for 15min at 4°C. 
8. Then the supernatant obtained was transferred into a new ice cold 50mL tube (Tube 2).
9. 1mL of 100% methanol was added to the original tube, then samples were homogenized using the same procedure.
10. Samples were centrifuged and supernatant transferred to the second tube.
11. Steps 9 and 10 were repeated twice.
12. The samples were left for 2 hours at -80°C in order to let any protein particles to precipitate.
13. Then NH4Ac-pH4.5 buffer was added to tube 2, in order to bring the final methanol concentration to 10%.

SPE-Washing

1. 500mg C18 SPE cartridges were conditioned.
   • Add one load (9.45mL) of methanol; leave methanol for 10 min.
   • Turn on the vacuum.
   • Then let the methanol go through SPE, and add a load (9.45mL) of NH4AC-pH4.5 buffer. Let the NH4AC-pH4.5 buffer for 10 min in the cartridge.
   • Then let the buffer go through the SPE and close it.
2. The 10% Methanol/ NH4AC-pH4.5 buffer solution was poured to the cartridge and then the vacuum manifold was opened.
3. The solution from above step 13 went through the SPE drop-wise.
4. When the solution was about to be finished the SPE was closed and the cartridges washed with NH4AC-pH4.5 buffer. Cartridges were washed once with NH4AC-pH5.5 buffer solution.
5. Then 1mL of 10% Methanol- NH4AC-pH4.5 buffer was added to each cartridge.
6. Then 0.5mL deionized water (18 Ω) was poured to each cartridge.
7. 1mL of Hexane was added to each cartridge, this in order to remove water and fat from the cartridge, if you need analyze DHA, avoid this step, just vacuum the cartridge for 5 min.
8. Vacuum the cartridges for 5 min.
9. The vacuum manifold tips were carefully dried with paper.

Elute Analytes out of SPE tubes

1. Turn off the vacuum.
2. Put 10ml coned glass tube under the cartridge. to collect MeOH fractions.
3. Add 1ml MeOH to each cartridge.
4. Add 9ml 20% MeOH/methyl formate (MF) to each cartridge.
5. Let solvent soak in the cartridge for about a minute before opening vacuum.
6. Open the vacuum gradually until 20% MeOH/MF (start to dripping slowly from the outlet of SPE tube

Nitrogen Drying

1. Use new glass tips
2. Place the needle or glass tips ~1cm above the liquid surface of the sample.
3. Dry down solvent gently till ~50 ul liquid left.
4. Using 100 µl METHANOL/each wash to wash lipids on the wall of 15ml-glass tube down to bottom, transfer methanol rinse into 500ul-vials
5. Repeat 3 times: 1st, wash the top of the 15ml-glass-tube; 2nd, wash middle of middle of 15ml-glass-tube; 3rd wash low end of 15ml-glass-tube into the bottoms, then transfer to 500ul-vials.
6. Make sure no deposit in the solution. If seeing deposits, the solution may need to be filtered with 0.2um nylon syringe-filter.
7. Dry down to final volume of 50ul.
8. Vortex all vials to make sure that the analytes are dissolved and solutions are homogenous. Ready for LC-MS analysis
9. Label each vial with #, date, a brief description or abbreviations, and total ul.

Protocol for Lipid Extraction from Skin Wounds

1. Samples were cut into small pieces. When cutting the samples, they were located on a plastic cap on ice.
2. The small pieces were transferred to a 5mL tube.
3. 1mL mixture of methanol and NH4AC-pH5.5 (MeOH-NH4AC-pH5.5=2:1) was added to each sample.
4. pH was checked and changed to a range of 4.5-5.5. pH was regulated by adding 1mL of Acetic acid.
5. 50µL of internal standard (PGD2-d4) with a concentration of 0.1ng/µL were added to samples.
6. Then the samples were homogenized using a probe sonicator. The tissues were homogenized for 1 minute (when homogenizing, the tubes were close to the ice).
7. After homogenizing samples were centrifuged at 5000rpm for 15min at 4°C.  
8. Then the supernatant obtained was transferred into a new ice cold 50mL tube (Tube 2).
9. 1mL of 100% ACETONE was added to the original tube, then samples were homogenized using the same procedure before discussed.
10. Samples were centrifuged and supernatant transferred to the second tube.
11. Steps 9 and 10 were repeated twice.
12. The samples were left overnight at -80°C; this in order to let any protein particles to precipitate.
13. Then NH4AC-pH5.5 buffer was added to tube 2, in order to bring the final acetone concentration to 10%.

SPE-Washing

1. 500mg cartridges were reconditioned.

• Add one load (9.45mL) of methanol; leave methanol for 10 min.
• Turn on the vacuum.
• Then let the methanol go through SPE, and add a load (9.45mL) of NH4AC-pH5.5 buffer. Let the NH4AC-pH5.5 buffer for 10 min in the cartridge.
• Then let the buffer go through the SPE and close it.

2. The 10% Methanol/ NH4AC-pH5.5 buffer solution was poured to the cartridge and then the vacuum manifold was opened.
3. The solution went through the SPE drop wise.
4. When the solution was about to be finished. The SPE was closed and the cartridges washed with NH4AC-pH5.5 buffer. Cartridges were washed once with NH4AC-pH5.5 buffer solution.
5. Then 1mL of 10% Methanol- NH4AC-pH5.5 buffer was added to each cartridge.
6. Then 0.5mL deionized water (18 Ω) was poured to each cartridge.
7. 1mL of Hexane was added to each cartridge, this in order to remove water from the cartridge.
8. Air passed through cartridges for 5 min.
9. The vacuum manifold tips were carefully dried with paper.
10. Cartridges were used 3 times before being disposed.

Elute Analytes out of SPE tubes

1. The SPE manifold top was moved to another glass manifold.
2. 10mL tips were attached to 250µL vials, under the cartridge.
3. 1mL of methanol was poured to each cartridge, to elute lipids.
4. 9mL of 10% MeOH/methyl formate were used to wash tubes and then this solution was poured to the cartridges.
5. The solution soaked into the cartridge for 1min before opening the vacuum.
6. Gradually, the vacuum was opened until the solution started dripping out; it must be done in a drop wise manner.
7. The solution ran through the SPE.

Nitrogen Drying

1. New glass tips were attached to the nitrogen dryer.
2. Nitrogen went through the tips in order to remove dust.
3. The tips were place at a level where nitrogen could blow the solution surface (about 1cm above the liquid surface).
4. Solvent was dried until it had a total volume of 100µL.
5. 200µL methanol was used to wash lipids from the 10mL tip’s wall.
6. The top of the tip was first washed
7. Then the middle area
8. At the end the lower tip area
9. The solution was dried to a final volume of 100µL.
10. The vials were vortexed; this to make sure that the analytes were dissolved and the solutions were homogenous.
11. 200 µL of H2O were added to the total volume.
12. Samples are kept in a storing box at -80°C.

Protocol for Lipid Extraction from db/db Diabetic and db+ Non-diabetic Macrophages

Incubation:

1. Prepare macrophages from mice following the published protocols.
2. Place the cells collected from mice in culture dishes.
3. Washed away non-attached cells.
4. Take pictures for attached cells, 3 fields were chosen for each dish
5. Incubated macrophages with 5ug DHA in 3ml PBS for each dish for 20min.
6. Then stimulate cells with 10ng/ml TNF-alpha, IL-1 beta, and 100ng/ml LPS for 1h.
7. Add 1ml ACN into dish to stop reaction and stored at -80 degree.
8. Calculate the cell number/field as cell ratio.

Lipid Extraction

1. To each sample, add 50 ng PGD2-d4 (1ng/ul)
2. Thaw the culture on ice.
3. Centrifuge at 5000rpm for 10min at 4°C
4. Transfer the supernatant into a ice-cold 50ml-tube (tube 2)
5. Add 0.25ml ACN into tube 1 and sonicate cells
6. Sonicate the culture on ice for 10seconds/pulse x 5.
7. Centrifuge at 5000rpm for 10min, transfer supernatant into tube 2
8. Repeat 6,7 for twice.
9. Add [NH4Ac-pH5 buffer] in tube 2 to bring the final concentration of ACN to 10%.
10. Go through SPE slowly (dropwise).

Washing

1. When liquid nearly runs out of SPE, turn off SPE, wash each cartridge with 3 loads [NH4Ac-pH5 buffer] to remove proteins.
2. When liquid nearly runs out of SPE, turn off SPE, wash each cartridge with half load of [10%MeOH,NH4Ac-pH5 buffer] to remove yellow staff.
3. Wash each cartridge with 0.5ml deionized-water(pH ~6)
4. 1ml hexane was used to take away water.
5. Let the air go through cartridge for 5min to dry water.
6. Using Kimwipes to sorb the water attached at the tip of the SPE tubes and flow outlet

Elute analytes out of SPE tubes

1. Turn off the vacuum
2. Put 10 ml TIP-ATTACHED-ON-500ul-VIALS under the cartridges to collect ACN/methyl formate fractions.
3. Add 1ml acetonitrile into SPE cartridge to elute lipids.
4. Add 9ml 10% ACN/methyl formate (MF) to each cartridge.
5. let solvent soak into cartridge for about a minute before opening vacuum
6. Open the vacuum gradually until 10% ACN/MF (start to dripping slowly from the outlet of SPE tube
7. Let 10% ACN/MF run through the SPE dropwise (i.e., slowly)

Nitrogen drying

1. Use new glass tips
2. Wash/rinse the 10ml tips with ~1-2ml of MeOH to remove the dust.
3. Place the needle or glass tips ~1cm above the liquid surface of the sample.
4. Dry down solvent gently till ~50 ul liquid left.
5. Using 100 µl  ACN/each wash to wash lipids on the wall of 10ml tip down into 500ul-vials
6. Repeat 3 times: 1^st, wash the top of the tip; 2^nd, wash middle of tip; 3^rd wash low end of tip into the 500ul-vials.
7. Make sure no deposit in the solution. If seeing deposits, the solution may need to be filtered with 0.2um nylon syringe-filter.
8. Dry down to final volume of 200ul.
9. Vortex all vials to make sure that the analytes are dissolved and solutions are homogenous. Ready for LC-MS analysis
10. Label each vial with #, date, a brief description or abbreviations, and total ul.