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

Abstract

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.

Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface

“…For instance, this protocol uses a methanol:acetone fixative for the staining of tight junction ZO-1 due to a lack of consistent staining with PFA fixation, as reported previously [ 22 ]. Overfixation with formaldehyde can cause autofluorescence, as excessive reagent reacts with amines in proteins to generate fluorescent products [ 12 ]. No noticeable autofluorescence was observed using the volume and duration of PFA incubation recommended in this protocol.…”

“…Immunofluorescence (IF) staining is the method of choice in examining the cell morphology and subcellular localization of proteins [ 12 ]. IF relies on the binding of a primary antibody to the target protein.…”

Glyoxal Fixation Is Optimal for Immunostaining of Brain Vessels, Pericytes and Blood-Brain Barrier Proteins

“…However, VE-cadherin staining showed a significant decrease in detection capacity in PFA fixed brain sections compared to glyoxal fixed brain sections. This difference in staining can be attributed to their nature of expression and antigen masking due to high crosslinking observed in PFA fixed brain [ 46 ]. To overcome antigen masking from the high crosslinking during PFA fixation, different antigen retrieval methods can be used, including high-temperature treatment in citrate buffer and enzymatic treatment with pepsin [ 33 , 46 , 47 ].…”

“…This difference in staining can be attributed to their nature of expression and antigen masking due to high crosslinking observed in PFA fixed brain [ 46 ]. To overcome antigen masking from the high crosslinking during PFA fixation, different antigen retrieval methods can be used, including high-temperature treatment in citrate buffer and enzymatic treatment with pepsin [ 33 , 46 , 47 ]. However, this adds time and cost to the procedure and this could also result in a non-uniform or inconsistent staining pattern due to differences in antigen exposure to retrieval methods.…”

Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA‐PAINT‐ERS

“…Basic Protocol 2 describes the steps needed for the preparation of unextracted U2OS or other (such as COS7) cell samples for DNA-PAINT-ERS imaging. However, some targets benefit from extraction of cells prior to immunostaining (Piña et al, 2022). In this protocol, the cells are pre-permeabilized with Triton X-100 before fixation.…”