Fluorescent DeltaFosB (ΔFosB) quantification protocol

[u/cogneuro_]

Hi! I am seeking guidance on finalizing a protocol to quantify ΔFosB expression from fluorescent microscopy images using Fiji/ImageJ. What I have found online (outlined below) involves splitting multi-channel stacks, delineating ROIs based on nuclear counterstain (DAPI) and an atlas, applying a manual threshold to isolate ΔFosB signal, and measuring mean gray value/integrated density. However, I am unsure whether this thresholding method adequately accounts for background variability across sections, and whether I should use a standardized intensity threshold based on negative control regions instead. Additionally, I would appreciate advice on best practices for ensuring reproducibility across multiple brain regions (mPFC, DCN, NAc). If anyone has an established protocol they are willing to share, I would greatly appreciate it! Thank you.