6 In the case of kidney glomeruli in adult rats, this would be about 25 μm 7,8 (at 5 slices with a thickness of 5 μm). The reason why they are separated 6 slices from their pair section (“volume section”) is because the optimal distance between these sections must be approximately 1/3 of the mean diameter of the structure to be measured (in this case, the glomerulus). Sections 106, 206, … are paired sections that will be used to estimate the numerical density (number of glomeruli/mm 3) according to the physical dissector method. It is important to obtain a minimal number of slices (greater than or equal to 6) needed to obtain a volume with an acceptable error coefficient. They are obtained systematically (there is always the same distance between the slices or T), uniformly (each of the slices are parallel) and randomly (the first slice is chosen arbitrarily between the slices 0 and T, with T being the same as 100 slices in this case). The sections 100, 200, … are to calculate the total volume of the kidney or the Cavalieri reference volume, and we will call them “volume sections”. If this happens with the slice that we are supposed to select, we will advance 5 μm or whatever is necessary to obtain an optimal slice, as long as we then maintain the distance of 25 μm with its corresponding paired section, and we continue with the initially established system. We will choose only those slices that are not broken or folded when laying them out on the slide. Another problem that may arise when tackling this objective is whether or not it is necessary for the renal cortex to be isotropic in order to apply the method and whether the size of the particles may lead to an error.Īs has been explained previously in classic articles, 5 that problem can be solved with adequate sampling, which gives each particle, in this case glomeruli, the same probability of being counted.įor the stereological study, we will divide the sections or slices number 100 and 106, 200 and 206, … and successively in the same manner until the end. Orientation does not influence the calculation of the volume and numerical density 1 (in our laboratory we make cross sections). We place a block with a kidney in the microtome and cut all of it into 5-μm thick sections. It is best to start with a pilot study that includes one or two animals, to gradually correct errors and adjust the steps (for example, how many fields we will use to measure glomeruli, how many dissectors per histological field, etc.). Once the blocks are made, they are kept in cabinets at room temperature.
Will undergo successive ethyl alcohol baths with increasing concentrations then they are cleared with xylol and embedded in paraffin (each kidney in a separate mould).
Rat kidneys are extracted and fixed for 24 h in 4% paraformaldehyde at room temperature. 2,3 Material and methods Sample preparation This will contribute to the resolution of many pathological, physiological, and biochemical problems.
#STEREOLOGY BASED COUNTING HOW TO#
Given the difficulty of stereological studies and the lack of a clear bibliography on their development, the objective of this work is to explain how to calculate the main stereological parameters of the kidney, step by step, in a simple manner and with examples utilising an animal model the idea is to establish easily understandable bases applicable in experimental studies. Stereology is a part of morphometry that allows three-dimensional information of cells or other biological structures to be collected from two-dimensional, serial, parallel, and equidistant microscopic slices by applying mathematical formulas based on statistically significant geometrical probability. In these cases, as well as under normal circumstances, stereological studies allow us to detect and quantify macro- or microscopic changes. The number, size, and distribution of nephron components (glomeruli, tubules, …) provide important information about renal function, making their quantification important in studies of traumas, chemicals and/or diseases. Kidneys are an essential organ in the maintenance of homeostasis, and nephrons are their structural and functional unit.