Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay.

Effects of chronic malnourishment and aging on the ultrastructure of pyramidal cells of the dorsal hippocampus.

OBJECTIVES: Malnourishment (M) produces permanent alterations during the development of the CNS and might modify the aging process. In pyramidal neurons (PN) of the hippocampus, which are associated with learning and memory performance, few studies have focused on changes at the subcellular level under chronic malnutrition (ChM) in young (Y, 2 months old) and aged (A, 22 months old) rats. The present work evaluated the extent to which ChM disrupts organelles in PN of the dorsal hippocampus CA1 as compared to controls (C). METHODS: Ultrastructural analysis was performed at 8000× and 20 000× magnification: Nucleus eccentricity and somatic, cytoplasmic, and nuclear areas were measured; and in the PN perikaryon, density indices (number of organelles/cytoplasmic area) of Golgi membrane systems (GMS, normal, and swollen), mitochondria (normal and abnormal), and vacuolated organelles (lysosomes, lipofuscin granules, and multivesicular bodies (MVB)) were determined. RESULTS: The density of abnormal mitochondria, swollen GMS, and MVB increased significantly in the AChM group compared to the other groups. The amount of lipofuscin was significantly greater in the AChM than in the YChM groups - a sign of oxidative stress due to malnutrition and aging; however, in Y animals, ChM showed no effect on organelle density or the cytoplasmic area. An increased density of lysosomes as well as nucleus eccentricity was observed in the AC group, which also showed an increase in the cytoplasmic area. DISCUSSION: Malnutrition produces subcellular alterations in vulnerable hippocampal pyramidal cells, and these alterations may provide an explanation for the previously reported deficient performance of malnourished animals in a spatial memory task in which aging and malnutrition were shown to impede the maintenance of long-term memory.