M205 fluorescence microscope

Brain vibratome sections were washed in 24-well plates with PBS, and sections were closed with 10 mL of 0.2% TritonX-100 and 150 μL of sheep serum protein for 2 h. Primary antibodies were incubated overnight at 4 °C. Primary antibodies included Iba−1 (1:100, AiFang, Changsha, China), GFAP (1:100, Proteintech, Wuhan, China), TNF−α (1:100, Proteintech, Wuhan, China), and IL−1β (1:100, ABclonal, Wuhan, China).
Sections were rinsed three times with PBS, incubated for 2 h at room temperature, and protected from light following the addition of secondary antibodies conjugated with Alexa Fluor 488 goat anti-rabbit (1:500, Beyotime, Shanghai, China) or goat anti-mouse cy3 (1:500, Beyotime, Shanghai, China). Sections were rinsed three times with PBS, attached to slides, dried in an oven at 37 °C for 5 min, and sealed with a sealer containing DAPI (SouthernBiotech, Birmingham, AL, USA). The images were observed and acquired using a Leica M205 fluorescence microscope. ImageJ (NIH, Bethesda, MD, USA) was used to count the percentage coverage of Iba-1 and GFAP in the area of positive staining in the field of view. The quantitative results concerning IL-1β and TNF-α were expressed as the cumulative fluorescence intensity in the field of view, and to intuitively observe the changes, the WTC group was used as a reference for normalized plotting.

Feeding RNAi plates harboring host bacteria HT115(DE3) engineered to express “quad” dsRNA (targeting pgl-1, pgl-3, glh-1, glh-4) were obtained from Susan Strome^{45 ,46 (link)}. L1 larvae were placed onto freshly prepared feeding RNAi plates with dsRNA induced by 1 mM IPTG (isopropyl-β-d(-)-thiogalactopyranoside) and were transferred after one generation at 25 °C and collected at F2 adults as described⁴⁵ for DAPI staining, oocyte and germline analysis.
For RNAi screen, L1 worms containing the his-72::mNeonGreen transgene were arrested in M9 media and then pipetted at a density of 20 worms per plate and maintained at 20 °C. At the L4 stage and during adulthood worms were scored for germline size using Leica M205 fluorescence microscope.
For the P granule germline immortality assay, three L1 worms were transferred every week on two replicate plates per condition at 20 °C. Plates were counted as sterile when no more than three worms were present after one week. Partial sterility was counted when one out of two plates became sterile while both replicate plates were sterile for full sterility.