An optimized method for Oil Red O staining with the salicylic acid ethanol solution

Figures & data

Figure 1. Solubility of ORO in different solvents. A: 15 ORO solutions show different degrees of red, representing different ORO dissolving abilities of 15 solvents. B. The absorbance at 492 nm of 15 ORO staining solutions semi-quantitatively reflects the solubility of ORO in different solvents. (n = 3. Error bars indicate mean ± SD. ns: p > 0.05, **P < 0.01; ****P < 0.0001, by ANOVA for multiple comparisons).

Figure 2. The staining effect of different dye solutions in different cells. A, C, E, G: Representative images of ORO staining for 143B human osteosarcoma cells (A), HSC-T6 rat hepatic stellate cells (C), APRE-19 human retinal epithelial cells (E), and adipocyte (G) adipocyte under the light microscope. Scale bars: 100 μm. B, D, F, H: The absorbance at 492 nm of ORO-containing extracts semi-quantitatively reflects the staining effect of 15 ORO staining solutions for 4 different cell lines. All the experiment was independently repeated three times. (n = 3. Error bars indicate mean ± SD. ns: p > 0.05, **P < 0.01; ***P < 0.001, ***P < 0.0001, by Student’s t-test between 2 groups comparisons).

Figure 3. The staining effect of different dye solutions for different tissue sections. A: Representative images with 15 kinds of staining solutions for continuous liver sections under the light microscope. Scale bars: 100 μm. B: Further staining with staining solutions of groups (2), (5), (8), and (9) for the aortic arch, skin, and adipose tissue sections. Scale bars: 200 μm. C: Quantitative comparison of the staining effect of groups (2), (5), (8), and (9) for the liver, aortic arch, skin, and adipose tissue sections. Representative of 3 photographs for every solution group and the mean was estimated. (n = 3. Error bars indicate mean ± SD. ns: p > 0.05, *: p < 0.05; **P < 0.01; ***P < 0.001, by ANOVA for multiple comparisons).

Table 1. Composition of different solvents for ORO preparation.

Group	Solvents	Formula (per 100 ml)
		100% isopropanol	100% ethanol	100% ethanol +20% salicylic acid	ddH2O
G1	100% isopropanol	100 ml	0 ml	0 ml	0 ml
G2	60% isopropanol	60 ml	0 ml	0 ml	40 ml
G3	100% ethanol	0 ml	100 ml	0 ml	0 ml
G4	70% ethanol	0 ml	70 ml	0 ml	30 ml
G5	50% ethanol	0 ml	50 ml	0 ml	50 ml
G6	50% ethanol+1% salicylic acid	0 ml	45 ml	5 ml	50 ml
G 7	50% ethanol+2% salicylic acid	0 ml	40 ml	10 ml	50 ml
G 8	50% ethanol+5% salicylic acid	0 ml	25 ml	25 ml	50 ml
G 9	50% ethanol+10% salicylic acid	0 ml	0 ml	50 ml	50 ml
G 10	30% ethanol	0 ml	25 ml	5 ml	70 ml
G 11	30% ethanol+1% salicylic acid	0 ml	30 ml	0 ml	70 ml
G 12	30% ethanol+2% salicylic acid	0 ml	20 ml	10 ml	70 ml
G 13	30% ethanol+5% salicylic acid	0 ml	5 ml	25 ml	70 ml
G 14	30% ethanol+10% salicylic acid	0 ml	30 ml	+ 10 g salicylic acid powder	70 ml
G 15	70% ethanol+10% salicylic acid	0 ml	20 ml	50 ml	30 ml

Data availability statement

The authors confirm that the raw data supporting the findings of this study are available within the article (and/or) its supplementary materials which can be accessed online at https://doi.org/10.1080/21623945.2023.2179334