Oil Red O Staining at the Lungs for Living Research Centre, UCL

by Charlotte Loos-Bennett

fig1While working in the Lungs for Living Research Centre at UCL I assisted with various experiments involving cellular biomedicine; particularly assessing ways to prevent the diminished function of the thymus (a lymphoid organ which produces T-lymphocytes for the immune system) with age. One said experiment was Oil red O staining.  The aim of this was to identify adipose tissue and distinguish between transgenic tissue (genetically modified or mutant type) and the wild type tissue (from individuals innatural conditions). The tissue for the slides was taken from a mammalian thymus.

For this experiment we were given 6 slides, four of which are from old thymus, and two test slides. Those from the old thymus (O series) acted as control slides and were expected to stain correctly. This is to represent how in the ageing process the thymus is infiltrated by adipose (fatty) tissue and undergoes thymus involution, where the thymus undergoes a progressive reduction in size leading to a reduced immune system. The other two slides (B series) were the test slides and were either a transgenic or a wild type. The slides had previously been fixed. The purpose of this was to make the tissue rigid.


Firstly, we rinsed the slides with 60% Isopropanol (300ml isopropanol, 200ml water) for 2 minutes before draining the slides. We then rinsed them with the Oil red O working solution (50% Oil red O with 50% water) for 15 minutes which would stain the adipose tissue red. Following this, a further rinse with Isopropanol was undertaken. To aid the rinsing of the slides they were placed on an orbital shaker. The slides were then dipped 5 times consecutively in the stain Haematoxylin (which stains nuclei blue) and rinsed in distilled water for 5 minutes. The final step in the staining process was to administer some Mowiol mount and cover the slide with a cover slip, ensuring there were no bubbles.

fig4When viewing, under the microscope and then using a Nanozoomer, each of the O series you could see how the adipose had infiltrated the thymus. For example O1 there was nothing but adipose tissue but O2, while there was still adipose; inside on half of the sample we observed blue (the stained nuclei). We concluded for O1 that you could only see red as when the slide had been cut; it had not gone deep enough into the thymus to get any of the actual tissue, as opposed to the adipose tissue on the surface. Once we had established the control we needed to identify which (from the B series) was the transgenic and which was the wild type. Upon observation we concluded it to be B2 that is the wild type. This is because B2 contained more blue and was therefore a much healthier tissue. B8 was a mutant as it was not packed with enough blue due to the lack of sufficiently healthy cells. While both samples contained adipose throughout it was the lack of healthy cells that hinted it was mutant and therefore fulfilled the aim of the experiment.