This automated protocol allows the histopathological preparation of murine tissues with robotic systems routinely used for processing and embedding of human samples, greatly increasing the quality of the samples. The main advantages of this technique, are that it reduces the number of methodological variables and it improves the standardization of procedural steps. Demonstrating the procedure, will be Francesca Boggio.
Francesca Boggio is a pathologist working in my laboratory. Begin by using small tweezers to transfer the fixed murine colon tissue samples from the neutral buffered formalin, onto a sectioning work plate in a Petri dish. Use a sterile scalpel to cut the colon into 0.2 to 0.3 centimeter fragments, and use the tweezers to place one segment into each of three non-adjacent plastic-protruding tips of an oriented paraffin-embedding cassette.
Carefully push the four edges to close the cassette and insert the grid into a plastic supporting frame. Then, label the supporting frame to identify the sample and transfer the cassette into the grid. One hour before processing, turn the automated processor.
When the instrument indicates that the paraffin has melted, manually insert the metal basket in the automated processor. When all of the cassettes have been inserted, close the basket and open the lid of the retort. Insert the basket into the dedicated housing of the processor and close the retort lid.
Use the touchscreen on the instrument computer to define the working protocol selecting the sequence of solutions, timing, and temperature, to be implemented according to the scheme provided in the table. Click on the dedicated computer icon to assign the protocol to the retort containing the cassette basket, and click the start button. When the instrument confirms the end of the protocol with a dedicated icon and an alarm tone, open the retort lid and remove the basket from the processor.
An hour before the tissue embedding, turn on the automated embedder and wait until the paraffin is completely melted as indicated by the paraffin bath thermometer on the instrument. Manually transfer up to 32 processed cassettes from the processor basket to the embedder rack and open the main embedder lid. Use the touchscreen to signal to the robotic system that a rack is being inserted and open the inlet housing lid.
Insert up to four racks into the inlet housing and close the inlet housing lid. Then, use the touchscreen to start the embedding procedure as indicated in the table. When the instrument confirms the end of the protocol with the dedicated icon, remove the outlet rack and close the main embedder lid.
Then, transfer the embedded blocks from the rack into a storage box. Colon shortening, and colonic expression of pro inflammatory genes, are widely used parameters for scoring the presence of dextran sodium sulfate or DSS induced inflammation. The infiltration of inflammatory cells into the intestinal lamina propria is also greatly enhanced by DSS treatment.
H&E staining of untreated samples processed by automation as demonstrated, reveals fewer infiltrating inflammatory cells, epithelial alterations, and changes in mucosal architecture compared to samples from DSS treated animals. The quality of the murine tissue preparation and embedding performed by the automated instruments can be additionally confirmed by immunohistochemical staining for CD20 and Mallory Trichrome Staining. Notably, automated sample processing consistently allows the evaluation of a higher proportion of histological parameters, than do samples processed by the manual method.
Take care to correctly insert the tissue into the grid to wait for the automated processor to be ready, and to wait for the paraffin to be completely melted. This method can be used to perform histological analysis of any murine tissue, with the qualitive of human histological samples which will be used in murine models of human pathologists. This technique has already been successful study for studying inflammatory tissue inflammation, and will be useful for studying models of colorectal murine cancer.
As some reagents used in this protocol are hazardous, remember to always wear protective gloves, goggles and a lab coat when performing this procedure.
