TITLE: HuBMAP | Formalin Fixation and Paraffin Embedding of Tissue Samples 
AUTHORS: Kelley   Knizner, Christopher   Simmons

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method details formalin fixation and paraffin embedding of the HuBMAP tissue specimens.</div><div class = "text-block"><span>Protocol adopted from </span><span style = "font-style:italic;">Marda Jorgensen, Jerelyn Nick (02/24/2020). HuBMAP: Paraffin Embedding Tissue Samples . </span><a href="https://dx.doi.org/10.17504/protocols.io.bam9ic96" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">https://dx.doi.org/10.17504/protocols.io.bam9ic96</span></a></div></div>

[STEPS]
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Processed cassette should be placed in wax holding chamber of embedding center.
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Pre-heat embedding utensils in the forceps warming station; warming chambers are located on the side of the paraffin dispensing nozzle.
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Turn on the station cold plate module.
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Open the cassette. Be sure to carefully maintain the tissue orientation. Dispose of cassette lid.
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Select an appropriate sized embedding mold.
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Partially fill a mold with molten paraffin by using forceps to tap the dispenser plate on the embedding center's dispensing nozzle.
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Carefully transfer tissue into the center of the mold using heated forceps. The tissue should be placed cut side down.
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Transfer the mold to the cold pad and gently press tissue flat with forceps, allowing the paraffin to solidify in a thin layer.
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Place the lower basket of the emptied cassette on top of the mold and add additional molten paraffin to fill 2/3 of the cassette basket. If there are bubbles, slowly lift and reposition the cassette to remove.
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Transfer the paraffin filled mold to the cold plate module and allow to solidify.
?. [TISSUE PROCESSING]
Tissue processing is done using the Leica Peloris II using the following program.Formalin30 minutesAmbient temperature75% Ethanol30 minutes37 degrees C80% Ethanol30 minutes37 degrees C95% Ethanol30 minutes37 degrees C95% Ethanol30 minutes37 degrees C100% Ethanol45 minutes37 degrees C100% Ethanol50 minutes37 degrees C100% Ethanol60 minutes37 degrees CXylene30 minutes37 degrees CXylene30 minutes37 degrees CXylene40 minutes37 degrees CXylene60 minutes37 degrees CParaffin30 minutes60 degrees CParaffin60 minutes60 degrees CParaffin90 minutes60 degrees C
Formalin30 minutesAmbient temperature75% Ethanol30 minutes37 degrees C80% Ethanol30 minutes37 degrees C95% Ethanol30 minutes37 degrees C95% Ethanol30 minutes37 degrees C100% Ethanol45 minutes37 degrees C100% Ethanol50 minutes37 degrees C100% Ethanol60 minutes37 degrees CXylene30 minutes37 degrees CXylene30 minutes37 degrees CXylene40 minutes37 degrees CXylene60 minutes37 degrees CParaffin30 minutes60 degrees CParaffin60 minutes60 degrees CParaffin90 minutes60 degrees C
?. During fixation, formalin will penetrate the tissue causing chemical and physical changes that harden and preserve the tissue's morphology and protect it against subsequent processing steps.Because melted paraffin wax is hydrophobic, most of the water in a specimen must be removed before it can be infiltrated with wax, so the tissue must go through dehydration in a series of ethanol solutions.Xylene is then used to clear the tissue,  displacing the ethanol which will be in turn displaced by molten paraffin wax.Because melted paraffin wax is hydrophobic, most of the water in a specimen must be removed before it can be infiltrated with wax, so the tissue must go through dehydration in a series of ethanol solutions.Xylene is then used to clear the tissue,  displacing the ethanol which will be in turn displaced by molten paraffin wax.