Effect of fixation and embedding methods on collagen fibrils diameter and spacing

Purpose The corneal tissue are usually fixed in fixatives and embedded in resin for ultrastructural studies. Here we study the effect of these fixatives and resins on the collagen fibril (CF) diameter and the spacing between the fibrils. Methods Four normal human post‐mortem corneal buttons of 24, 25, 54 and 75 year old individuals were used. A part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C under UV light for 24hrs. A second part of each cornea was fixed in glutaraldehyde (2.5%) + osmium tetroxide. The third part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for ultrastructural studies. The tissues were embedded in spurr resin at 70°C for 12 hrs. Ultra‐thin sections were stained with uranyle acetate and lead citrate. Results In the tissue, fixed paraformaldehyde (4%) and embedded in LR White at 4°C under UV light for 24hrs, the CF diameter was 24± 0.1nm and spacing between CF was 39±0.1nm. In the tissue fixed in glutaraldehyde (2.5%) + osmium tetroxide, and embedded in spurr resin, the diameter was 28.37 ± 5.84nm and spacing between CF was 45 ±4.57nm. In the tissue, fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr resin, the diameter was 33.75 ± 3.65nm and spacing between CF was 48.06 ±4.35nm. Conclusion Our study shows that there is a variation in the collagen fibril diameter and spacing depending on the method of fixation and embedding resins. This needs to be considered when comparative studies employing differ