Tissue Collection and Histology All animal tissues for protein or RNA extraction were frozen at time of collection unless otherwise stated and later ground to a fine powder using a sterile pestle and mortar on liquid nitrogen.
Mycelium was then scrapped off the surface of the plates using a sterile glass slide, immediately frozen in liquid nitrogen, and lyophilized overnight.
The ticks were ground in liquid nitrogen using a sterile mortar and pestle and total RNA was isolated using the TRIzol® reagent according to the manufacturer's protocol (GibcoBRL, USA).
All these samples were ground in liquid nitrogen using a sterile mortar and pestle, and then the RNA was isolated using the mirVana microRNA isolation kit according to manufacture's instructions (Ambion, Applied Biosystems).
Second, after foaming, the cement powder was incorporated in the liquid foam and further mixed using a sterile spatula, preventing foam disruption, until complete homogenization of the paste.
Follicular fluid was sampled from all the mature follicles of both ovaries using a sterile needle and syringe, put into cryo-tubes, plunged into liquid nitrogen and stored at −80°C until analyzed for cytokine concentrations.
Spirogyra pratensis filaments were removed from the medium using a sterile glass hook, wicked to remove excess moisture, dropped in liquid nitrogen and stored at -80°C until RNA extraction.
Thereafter, the mucosa was collected by scraping using a sterile glass microscope slide at 4°C [ 26, 38], rapidly frozen in liquid nitrogen, and stored at −80°C until analysis.
After rinsing in DEPC-treated water, the adult ticks were frozen in liquid nitrogen and subsequently crushed and ground to powder using a sterile mortar and pestle.
Nematode infected root tissue was excised using a sterile scalpel at 3 days post-inoculation (dpi) and 9 dpi, respectively, under a magnifying glass and flash frozen immediately in liquid nitrogen.
