Axioplane 2

Only cell profiles with clear focal plane outlines were included in the analysis. The optical densities (ODs) of immunohistochemical staining products in the tissue sections were obtained with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). A digital camera, mounted on Zeiss microscope (Axioplane 2, Carl Zeiss MicroImaging GmbH, Hamburg, Germany), was used to image the sections at 50× magnification in bright field, which were displayed on a high-resolution monitor. All OD readings from cells in each section were combined and averaged to obtain the total OD (TOD) of each section. An average of ten random rectangles (rectangular area = 150 μm²) in the lumen of the blood vessel were used to measure the background staining (BOD) of each region. By subtracting BOD from TOD, the true OD for each part of the background correction was obtained. For uniform settings, all images were captured on the same day. All parameters were carefully adjusted according to recommended gray levels, histogram stretching, and minimum OD. The Nissl straining was observed using a Zeiss microscope (Axioplane 2, Carl Zeiss MicroImaging GmbH, Hamburg, Germany), and these results were quantified using the Image J software (NIH, Bethesda, MD, USA).

Air dried thin smears of P. berghei ookinete or schizont or sporozoites and oocysts, obtained from midgut of P. berghei infected Anopheles stephensi crushed in PBS, were fixed with 4% EM grade paraformaldehyde (Electron Microscopy Science) for 10 minutes. Permeabilisation, blocking and incubation with primary and appropriate secondary antibodies (S4 Table & S5 Table) was performed as described by [41 (link)] with additional last washes with 70% ethanol and absolute ethanol 1 min each, air-dried and mounted in VectaShield (Vectorlabs) containing DAPI (4', 6-diamidino-2-phenylindole) in glycerol for nuclear staining. Parasites were examined either under Delta Vision Epifluorescence microscope (Applied Precision). 100x objective, images were captured with CoolSNAP HQ camera (Photometrics) and deconvoluted using SoftWoRx software (Applied Precision) or under Axioplane2 (Zeiss) 100x objective, images were taken through HAMAMATSU ORCA_ER camera (HAMAMATSU) and Velocity software 4.1.0 (PerkinElmer). Images were processed using Fiji (NIH) as well as SoftWoRx explorer 1.3. Super-resolution images were captured through Elyra PS.1 super-resolution microscope (Zeiss) under 60x objective with sCMOS PCO camera and images were visualized with ZEN Black software (Zeiss) and processed with ZEN LITE software (Zeiss).

The computerized image analysis for quantifying the expression pattern of neurochemicals was carried out in those animals subjected to immunohistochemistry (n = 18). The general approach for quantitative image analysis was similar to our previous studies [68 (link),71 (link)]. A computer based image analysis system along with the Image-Pro Plus software 6.0 (Media Cybernetics, Silver Spring, MD, USA) was used to quantify the amount of positive labeling. A digital camera mounted on a ZEISS microscope (Axioplane 2, Carl Zeiss MicroImaging GmbH, Hamburg, Germany) imaged sections and displayed them on a high-resolution monitor. A total of 20 sections per animal, representing the entire rostro-caudal extent of the CeA, were analyzed. The number of CeA neurons (or nuclei) reacted for CRF, SP, and c-fos was then densitometrically counted, and all readings were combined and averaged to obtain the total amount of labeling. For quantifying the morphological changes of the microglia (ranging from simple rounded to complex branched), the value of fractal dimension (D) was used as an indicator in which a higher D means a greater complexity of the expression pattern [72 (link)]. All images were captured on the same day by the same experimenter to maintain the uniform settings adjusted at the beginning of capturing.