Ultrapure low melting agarose

Adult female Anopheles stephensi mosquitoes were killed at various times following feeds on mice infected with P. berghei, and fixed overnight at 4°C in a 1:1 mixture of 4% paraformaldehyde in 1x PBS and 100% ethanol. Mosquitoes were then washed 3 times in 1xPBS for 5 minutes each time. Washed mosquitoes were embedded in 1.3% ultrapure low-melting agarose (Invitrogen) in deionized water. Gels containing the mosquitoes were transferred for at least 2h to 4°C. Using a single-edge blade, the gel was then trimmed into a block containing a single mosquito in the centre.

Epithelial cells were extracted from pregnant mammary glands for RUNX2 western. Runx2^−/− mouse embryonic fibroblasts (Rx2KO MEFs) (Kilbey et al., 2007 (link)) were used as negative control. MDA-MB-231 was purchased from American Type Culture Collection. MDA-MB-231 cells were transfected with RUNX2 shRNA and scrambled control (HuSHTM, Origene) through electroporation using Nucleofector Kit V program X-013 (Amaxa, Lonza). For immunohistochemistry, cells were fixed with 2% paraformaldehyde for 15 minutes. After centrifugation, pellets were resuspended in 200 μl of 3% UltraPure^™ low-melting agarose (Invitrogen) and left for 20 minutes at RT to solidify. The agarose plug in 70% ethanol was embedded in paraffin blocks and stained as above.