Oil red O staining solution and Oil red O staining method

Abstract

The present invention discloses an Oil Red O staining solution composed of 0.5% Oil Red O, 50% ethanol, and 5%-10% salicylic acid. The staining solution does not contain any toxic organic solvents, is non-toxic, and has good safety. The present invention also discloses a method for staining cells or tissues with Oil Red O. The method has the advantages of simple operation, short staining time, easy staining of neutral fats in cells or tissues, good staining effect, and a clean background. The method does not use isopropyl alcohol, is safe and convenient, and is suitable for promotion and application in various laboratories.

Description

Technical Field

[0001] The invention belongs to the technical field of intracellular lipid staining, and particularly relates to an Oil Red O staining solution and an Oil Red O staining method. Background Art

[0002] Oil Red O staining is a method for visualizing fat in tissues or cells and is commonly used in pathological diagnosis and scientific research. Oil Red O is an oil-soluble azo dye, a strong lipid solvent and stain. It is highly soluble in fat and can specifically stain neutral lipids such as triglycerides and lipoproteins within cells or tissues. The principle is that Oil Red O is more soluble in neutral lipids than in the dye solution. During staining, Oil Red O transfers from the dye solution medium to the tissue fat medium, staining the intracellular lipid droplets red. Factors influencing the Oil Red O staining effect are: 1. The Oil Red O content in the dye solution; 2. The difference in Oil Red O solubility between the dye solution medium and the tissue fat medium. The greater the difference, the faster the transfer and penetration rate, and the greater the amount of Oil Red O that penetrates and accumulates within the tissue fat.

[0003] The preparation of the Oil Red O staining solution is crucial to its effectiveness. Oil Red O is readily soluble in benzene and soluble in organic solvents such as chloroform, glacial acetic acid, ether, ethanol, acetone, petroleum ether, fixed oils, hot glycerin, and volatile oils, but insoluble in water. Organic solvents are volatile and harmful to the human body, and precipitation is easily produced after the dye solution is prepared. This can easily leave red crystals on tissue sections during staining, affecting the interpretation of the results. These organic solvents themselves also dissolve fat in tissues, necessitating the addition of a certain proportion of water to the stock solution to prepare the solvent. This reduces its concentration and fat-dissolving capacity, preventing the dissolution of fat in the tissue, and increases the difference in solubility of Oil Red O between the dye solution medium and the tissue fat medium.

[0004] A 100% isopropanol-saturated Oil Red O solution is convenient for storage and is the most commonly commercially available reagent. This method, which is prepared with 60% isopropanol for staining, is convenient and provides excellent staining results, making it the most commonly used method for preparing Oil Red O staining solutions. However, as an organic solvent, isopropanol is volatile, and inhalation of isopropanol during use is harmful to the human body. Oil Red O is highly soluble in 60% isopropanol, resulting in a dark staining background and poor staining performance for samples with a small number of intracellular lipid droplets or smaller lipid droplets.

[0005] Ethanol is a non-toxic organic solvent that can be used for Oil Red O staining and is harmless to the human body. There are reports that 50% ethanol is used to prepare Oil Red O staining solution. However, the solubility of Oil Red O in 50% ethanol is not high, the staining effect is poor, and the lipid staining is light. Under the same conditions, the staining effect is far inferior to the Oil Red O staining solution prepared with 60% isopropanol.

[0006] Salicylic acid, with the molecular formula C7H6O3, is a fat-soluble organic acid soluble in organic solvents such as ethanol, acetone, and ether. It is commonly used as a key raw material in fine chemicals such as pharmaceuticals, fragrances, dyes, and rubber additives. Salicylic acid has anti-inflammatory and bactericidal properties, and 2-3% salicylic acid ethanol (containing 70% ethanol) is clinically used to inhibit bacteria, inhibit enzymes, and relieve itching. Salicylic acid also dissolves oil in pores, and 2% to 10% salicylic acid ethanol (containing 70% ethanol) is clinically used to exfoliate, shrink pores, and remove blackheads and blackheads, without any toxic effects on the human body. Summary of the Invention

[0007] In order to solve the problems that the current Oil Red O staining solution has toxic effects, a dark background of Oil Red O staining, and poor staining effect on small lipid droplets, the present invention provides an Oil Red O staining solution and an Oil Red O staining method.

[0008] This is achieved specifically through the following technical solutions:

[0009] An Oil Red O dye solution comprises 0.5% Oil Red O (mass fraction), 50% ethanol (volume fraction), and 5%-10% salicylic acid (mass fraction).

[0010] The present invention also provides a cell Oil Red O staining method, comprising the following steps:

[0011] Step 1: The cells to be tested are seeded into a culture dish or plate, and the treatment factors are added until the test time point;

[0012] Step 2: Aspirate the culture medium, wash 1-2 times with PBS, and fix with 4% paraformaldehyde for 20-30 minutes;

[0013] Step 3: Aspirate and discard the fixative; wash 3 times with PBS;

[0014] Step 4: Prepare the above-mentioned Oil Red O dye solution, filter it through filter paper or a 0.45 μm filter, and set aside;

[0015] Step 5: Aspirate and discard PBS, then add 50% ethanol for 15-20 seconds.

[0016] Step 6: Aspirate and discard 70% ethanol, add Oil Red O staining solution from step 4, and incubate at room temperature for 10-15 minutes;

[0017] Step 7: Aspirate and discard the Oil Red O staining solution, add 50% ethanol and differentiate for 15-20 seconds; wash with PBS three times until the liquid is clear, and photograph under a microscope;

[0018] Step 8: Aspirate and discard PBS, add 100% ethanol to dissolve Oil Red O, add 50 μl of ethanol solution to a 96-well plate, and detect the OD value at a wavelength of 492 nm using a microplate reader.

[0019] Preferably, the above cells are mammalian cells containing neutral lipids.

[0020] The present invention also provides a tissue Oil Red O staining method comprising the following steps:

[0021] Step 1: For tissues containing neutral lipids (liver, kidney, fat, aorta), frozen sections were prepared immediately after sampling, with a thickness of 12 μm.

[0022] Step 2: Fix the sections with 4% paraformaldehyde for 10 minutes, wash with distilled water for 5 minutes, and then rinse with 70% ethanol for 15-20 seconds.

[0023] Step 3: Prepare the above-mentioned Oil Red O dye solution, filter it through filter paper or a 0.45 μm filter, and set aside;

[0024] Step 4: Dye with Oil Red O solution for 10-15 minutes;

[0025] Step 5: Differentiate with 70% ethanol until the interstitial tissue is clear, and then wash with distilled water for 5 minutes;

[0026] Step 6: Counterstain with hematoxylin for 3-5 minutes and wash with distilled water for 5 minutes;

[0027] Step 7: Seal the slides with glycerol gelatin and observe under a microscope.

[0028] Technical Effects

[0029] 1. The present invention uses salicylic acid ethanol solution to prepare Oil Red O, which has no toxic effect on the human body.

[0030] 2. The Oil Red O staining method of the present invention is easy to operate, has a short staining time, and a clean staining background. The staining effect of the Oil Red O stain prepared with 50% ethanol + 5-10% salicylic acid as the solvent is comparable to that of the Oil Red O stain prepared with 60% isopropanol, and is even better than that of the Oil Red O stain prepared with 60% isopropanol.

[0031] 3. The Oil Red O solution prepared with salicylic acid ethanol solution can be stored for a long time (more than half a year), which is convenient for preparing Oil Red O staining kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Oil Red O dye solutions prepared for different solvents:

[0033] (1) 100% isopropyl alcohol; (2) 60% isopropyl alcohol; (3) 100% ethanol; (4) 70% ethanol; (5) 50% ethanol; (6) 50% ethanol + 1% salicylic acid; (7) 50% ethanol + 2% salicylic acid; (8) 50% ethanol + 5% salicylic acid; (9) 50% ethanol + 10% salicylic acid; (10) 30% ethanol; (11) 30% ethanol + 1% salicylic acid; (12) 30% ethanol + 2% salicylic acid; (13) 30% ethanol + 5% salicylic acid; (14) 30% ethanol + 10% salicylic acid; (15) 70% ethanol + 10% salicylic acid;

[0034] Figure 2 The OD values of Oil Red O dye prepared with different solvents at a wavelength of 492 on a microplate reader;

[0035] Figure 3 Oil Red O staining solutions prepared with different solvents were used to stain 143B human osteosarcoma cells (400×);

[0036] Figure 4 for Figure 3 The OD value of the cell after the oil red O was eluted at a wavelength of 492 on a microplate reader;

[0037] Figure 5 Oil Red O dye prepared with different solvents was used to stain HSC-T6 rat hepatic stellate cells (400×);

[0038] Figure 6 for Figure 5 The OD value of the cell after the oil red O was eluted at a wavelength of 492 on a microplate reader;

[0039] Figure 7 Oil Red O dye prepared with different solvents was used to stain APRE-19 human retinal epithelial cells (200×);

[0040] Figure 8 for Figure 7 The OD value of the cell after the oil red O was eluted at a wavelength of 492 on a microplate reader;

[0041] Figure 9 Staining of mouse liver tissue sections with Oil Red O dye prepared with different solvents (400×): (2) 60% isopropanol; (8) 50% ethanol + 5% salicylic acid; (9) 50% ethanol + 10% salicylic acid. DETAILED DESCRIPTION

[0042] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the above invention content.

[0043] Example 1.

[0044] Preparation of Oil Red O dye solution

[0045] 1) 0.5% saturated Oil Red O isopropanol and ddH2O were mixed at a ratio of 3:2 to prepare 60% Oil Red O isopropanol for staining as the control group.

[0046] 2) Prepare several 50ml centrifuge tubes and add 0.2g of Oil Red O powder to each tube (if there is a deviation in the weighing, 100ml of solvent can be added for every 0.5g of the actual Oil Red O mass to prepare a 0.5% Oil Red O solution).

[0047] Weigh 20 g of salicylic acid and dissolve it in 60 ml of ethanol and 40 ml of ddH2O to prepare a 20% salicylic acid ethanol (containing 60% ethanol) solution.

[0048] Prepare the solvent according to the table below (per 10 ml):

[0049]

[0050] Add 40 ml of the above solvents to the centrifuge tubes to prepare 0.5% Oil Red O solution, pipette evenly, and let it stand for 10 minutes. The dissolution of Oil Red O is as follows: Figure 1 As shown, the solubility observed by naked eye from large to small is as follows:

[0051] (1)=(3)=(15)>(9)=(2)>(8)>(4)>(7)=(14)>(6)>(5)>

[0052] (13)>(12)>(11)>(10).

[0053] At the same ethanol concentration, the solubility of Oil Red O increases with increasing salicylic acid concentration. The 30% ethanol + 5% salicylic acid and 30% ethanol + 10% salicylic acid groups produced flocculent precipitates, representing undissolved salicylic acid. Adding ethanol to a concentration above 50% dissolved the flocculent precipitates.

[0054] The above Oil Red O solution was filtered with filter paper or 0.45 μm filter, 50 μl was taken and placed in a 96-well plate, and the OD value at a wavelength of 492 nm was detected by microplate reader. The results (Table 1, Figure 2 )as follows:

[0055] Group 1 group 2 groups 3 groups 4 groups 5 groups OD492 OVERFLW 1.625±0.226 OVERFLW 1.5645±0.203 0.367±0.061 Group 6 groups 7 groups 8 groups 9 groups 10 groups OD492 0.635±0.046 0.832±0.037 1.821±0.039 2.679±0.107 0.021±0.002 Group 11 groups 12 groups 13 groups 14 groups 15 groups OD492 0.037±0.001 0.075±0.001 0.083±0.009 0.569±0.138 OVERFLW

[0056] Example 2.

[0057] Comparison of the staining effects of Oil Red O dye prepared with different solvents on 143B human osteosarcoma cells

[0058] Cell preparation: 143B human osteosarcoma cells were seeded in 48-well plates and stained after 24 hours of attachment.

[0059] Cell fixation: Aspirate the culture medium, add 500 μl PBS to each well, wash 1-2 times, add 4% paraformaldehyde to fix for 20-30 minutes; aspirate the fixative; add 500 μl PBS to each well, wash 3 times.

[0060] Oil Red O staining: Aspirate and discard PBS from the well plate, add 500 μl of 50% ethanol, wash for 15-20 seconds to remove moisture; add 200 μl of different Oil Red O stains filtered in Example 1 to each well and incubate at room temperature for 10-15 minutes; aspirate and discard the Oil Red O stain, add 500 μl of 50% ethanol, wash for 15-20 seconds to remove excess stain; wash with PBS three or more times until the liquid is clear, and photograph under a microscope.

[0061] The above staining was performed in triplicate, and groups (1) and (2) were washed and differentiated with 60% isopropanol.

[0062] Among them, 30% ethanol + 5% salicylic acid and 30% ethanol + 10% salicylic acid groups have flocculent precipitates, which are undissolved salicylic acid. Centrifugation or filtration can remove the flocculent precipitates without affecting the staining results of Oil Red O. Adding ethanol to a concentration of 50% or more can dissolve the flocculent precipitates. The Oil Red O dye solution with 70% ethanol + 10% salicylic acid and 100% ethanol as solvents has high solubility of Oil Red O and a bright red color equivalent to 100% isopropanol solution, but the staining effect is poor and it can hardly stain the lipids in the cells. Under the same washing conditions, a light red color can still be seen in the background ( Figure 3 : 1 group, 3 groups, 5 groups).

[0063] Except for groups 1, 3, and 15, the oil red O staining solution in other groups could stain lipid droplets; the lipid droplets in groups 10, 11, 12, and 13 were lightly stained, showing a light yellow color; the lipid droplets in other groups were orange-red to dark red. In groups (8) and (9), red staining of lipid droplets in the cytoplasm was visible after 5 minutes of staining. The lipid droplet staining effect was optimal when staining for 10-15 minutes. The red color of lipid droplets was no different from that of lipid droplets stained for more than 2 hours or even overnight (12-16 hours). The staining background was clean, without impurities or red crystals.

[0064] The Oil Red O staining effect under light microscopy is as follows Figure 3 As shown, the staining area and lipid droplet color from dark to light are: (2) = (9) = (8) > (7) > (6) = (14) = (4) > (5) > (13) > (12) > (11) > (10) > (1) = (3) = (15).

[0065] Aspirate and discard PBS (you can use a cotton swab dipped in 50% ethanol to wipe the sidewalls of the culture plate to remove excess Oil Red O staining solution), add 100 μl of 100% ethanol, incubate at room temperature for 10 minutes to dissolve the Oil Red O in the lipid droplets, draw 50 μl of ethanol solution into a 96-well plate, and place the 96-well plate in a 492 nm wavelength to detect the OD value ( Figure 4 ).

[0066] In summary, the 50% ethanol + 10% salicylic acid group (9) and the 50% ethanol + 5% salicylic acid group (8) had the best Oil Red O staining effect, which was equivalent to the 60% isopropanol group (2), and the staining of small lipid droplets in groups (9) and (8) was clearer than that in group (2); followed by the 50% ethanol + 2% salicylic acid group (7).

[0067] Example 3

[0068] Comparison of the staining effects of Oil Red O dye prepared with different solvents on HSC-T6 and APRE-19 cells HSC-T6 rat hepatic stellate cells were seeded in 48-well plates with an initial cell density of 15-20%. After the cells adhered, the culture medium was replaced with DMEM complete medium containing 100 μmol / L sodium oleate and 10 μmol / L all-trans retinoic acid for 5-7 days.

[0069] APRE-19 human retinal epithelial cells were seeded into 48-well plates with an initial cell density of 20-30%. After the cells adhered, they were replaced with DMEM / F12 complete medium containing 100 μmol / L sodium oleate for 48 hours.

[0070] The staining results of HSC-T6 rat hepatic stellate cells were shown in the above oil red O staining solution. Figure 5 and Figure 6 ; The staining results of APRE-19 human retinal epithelial cells are shown in Figure 7 and Figure 8 The staining effect trend of Oil Red O dye prepared with different solvents was the same as that of 143B human osteosarcoma cells. The 50% isopropanol + 10% salicylic acid group (9) and the 50% ethanol + 5% salicylic acid group (8) had the best effect, which was equivalent to or even exceeded the 60% isopropanol group (2), followed by the 50% ethanol + 2% salicylic acid group (7).

[0071] In addition to the above cells, other mammalian cells containing neutral lipids can be stained with Oil Red O using this method.

[0072] Example 4.

[0073] Staining effect of modified Oil Red O stain on liver tissue sections

[0074] Fresh livers were obtained from mice with an 8-week CCL4-induced liver injury model and frozen sections (12 μm thick) were prepared. The sections were fixed with 4% paraformaldehyde for 10 minutes, washed thoroughly with distilled water for 5 minutes to remove the formaldehyde, and then washed with 70% ethanol (the control group was washed with 60% isopropanol). The sections were stained with Oil Red O (50% ethanol + 10% salicylic acid and 50% ethanol + 5% salicylic acid, and 60% isopropanol as a control) for 10-15 minutes. The sections were differentiated with 70% ethanol until the interstitial structure was clear (the control group was differentiated with 60% isopropanol), washed thoroughly with distilled water for 5 minutes, counterstained with hematoxylin for 3-5 minutes, and washed with distilled water for 5 minutes. The sections were mounted with glycerol-gelatin and observed under a microscope.

[0075] Oil Red O staining effect is shown Figure 9 The liver tissue background of the 50% ethanol + 10% salicylic acid group (9) and the 50% ethanol + 5% salicylic acid group (8) was clean, without red crystals and impurities. The lipid droplets varied in size and were evenly distributed in the liver cytoplasm. The cell nuclei were clearly stained. In the 60% isopropanol group (2), a few purple-red to red-black impurities and red lipid droplets floating on the cell nuclei were visible. The lipid droplets of the 50% ethanol + 10% salicylic acid group (9) and the 60% isopropanol group (2) were dark red, while the lipid droplets of the 50% ethanol + 5% salicylic acid group (8) were orange-red. In addition, the number of small red lipid droplets in the liver cells of the 50% ethanol + 10% salicylic acid group (9) was more than that of the 60% isopropanol group (2).

[0076] In addition to the liver, other tissues containing neutral lipids such as kidney, fat, aorta, etc. can be stained using this method.

Claims

1. An Oil Red O dye solution comprising 0.5% by mass of Oil Red O, 50% by volume of ethanol, and 5% to 10% by mass of salicylic acid.

2. A cell Oil Red O staining method comprising the following steps: Step 1: The cells to be tested are seeded into a culture dish or plate, and the treatment factors are added until the test time point; Step 2: Aspirate the culture medium, wash 1-2 times with PBS, and fix with 4% paraformaldehyde for 20-30 minutes; Step 3: Aspirate and discard the fixative; wash 3 times with PBS; Step 4: Prepare the Oil Red O dye solution as described in claim 1, filter it with filter paper or 0.45 μm filter and set aside; Step 5: Aspirate and discard PBS, then add 50% ethanol for 15-20 seconds. Step 6: Aspirate and discard 70% ethanol, add Oil Red O staining solution from step 4, and incubate at room temperature for 10-15 minutes; Step 7: Aspirate and discard the Oil Red O staining solution, add 50% ethanol and differentiate for 15-20 seconds; wash with PBS three times until the liquid is clear, and photograph under a microscope; Step 8: Aspirate and discard PBS, add 100% ethanol to dissolve Oil Red O, add 50 μl of ethanol solution to a 96-well plate, and measure the OD value at a wavelength of 492 on a microplate reader.

3. The dyeing method according to claim 2, further characterized in that: The cells are mammalian cells containing neutral lipids.

4. A tissue Oil Red O staining method comprising the following steps: Step 1: For tissues containing neutral lipids, prepare frozen sections immediately after sampling, with a thickness of 12 μm; Step 2: Fix the sections with 4% paraformaldehyde for 10 minutes, wash with distilled water for 5 minutes, and then rinse with 70% ethanol for 15-20 seconds. Step 3: Prepare the Oil Red O dye solution as described in claim 1, filter it with filter paper or 0.45 μm filter and set aside; Step 4: Dye with Oil Red O solution for 10-15 minutes; Step 5: Differentiate with 70% ethanol until the interstitial tissue is clear, and then wash with distilled water for 5 minutes; Step 6: Counterstain with hematoxylin for 3-5 minutes and wash with distilled water for 5 minutes; Step 7: Seal the slides with glycerol gelatin and observe under a microscope.

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