Questions regarding paraffin embedding of spheroids:

• How can spheroids be embedded into paraffin?
  Spheroids are harvested (using standard pipette tips, where the tip was cut away [to enlarge the hole]) in 15ml tubes (at least 96 spheroids per experimental group) and centrifuged (3min, 200-500g). Subsequently, the supernatant is removed and the spheroid pellet is fixed for 12-24h (4°C) in 4% freshly prepared parafomaldehyde in HBSS or PBS. Centrifuge the spheroids again and remove the fixative. Wash the spheroids in water (10ml per tube) for 30min. Centrifuge again, remove the supernatant, suspend the spheroids in 70% ethanol and incubate for 45-60min (room temperature). Repeat this step with 85% ethanol, 96% ethanol and isopropanol. Remove isopropanol exept for 1,5ml. Carefully transfer the spheroid pellet together with 1ml isopropanol in a 1,5ml safe lock cup. Let the spheroids settle down and remove the supernatant. Fill the cup with 1,4ml 65°C temperatured low melting paraffin (melting temperature: 44-48°C), close the cup and place it into an oven (65°C) for 15min. Invert the cup several times to suspend the spheroids in the paraffin. Make sure that the tip of the cup is oriented towards the bottom and incubate it for 12h at 65°C. Place the tip of the cup into ice cold water till the paraffin becomes solid (the spheroids will be trapped in the solid phase) and discard the supernatant (note that only a small amount of low melting paraffin (ca 50µl) should remain in the cup. Otherwise it will disturb the solidification of high melting paraffin). Fill the cup with 1,4ml 65°C temperatured high melting paraffin (paraplast, melting temperature:56-58°C) and incubate it for 15min at 65°C. Invert the cup several times to suspend the spheroids. Because high melting paraffin will solidify rapidly during this procedure, make sure that all of the spheroids become suspended before the paraffin becomes solid. Orientate the tip of the cup towards the bottom and incubate it for 12h-24h at 65°C. Thereafter let the paraffin cool down to room temperature. Cut away the tip of the cup using a scalpel. If you carefully squeeze the cut tip, you easily can remove the paraffin plug inside with a tweezer. The spheroids are located at the front of the tip. Embed the paraffin plug frontside up in a paraffin block. Because the spheroids are located only in a thin layer inside the upper part of the paraffin block, control the paraffin sections using a microscope during sectioning to ensure that you don´t cut away the spheroids.
  
  
• Is it possible to embed collagen gels in paraffin to analyze sprouts originating from the spheroids?
  It is possible but it will take several days (70%, 85%, 96% Ethanol each at least 12-24h, Isopropanol 12-24h, paraffin I (low melting) 24h, paraffin II (high melting) 24-48h). Another problem is (depending on the number of embedded spheroids) that you have to make a lot of sections to hit one or more spheroids/sprouts. Note that fixation of collagen gels in formaldehyd-containing fixatives makes the collagen more fragile and friable. For confocal imaging it is advisable to use whole mount protocols for immunhistochemistry and analysis or to exchange water in the matrix against glycerin. Collagenase will disrupt the entire structure of the matrix and most of the sprouts might be destroyed.