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.

Keywords: antibody; immunocytofluorescence; immunohistofluorescence; immunolabeling; immunolocalization; signal-to-noise ratio; unmask epitopes.

Figure 1

The immunostaining method and its common disadvantages through the metaphor of a highway: Vertical white arrow displays routine steps of the indirect immunolabeling method (The Immunostaining Route); after every stage some usual drawbacks (Roadblocks) related to each step are represented with numbers (Way to) and described in the road signs of roadblocks (left). Some of these technical disadvantages are tackled by the methodologies (Detours) presented in this review and represented with the corresponding number (Exit). Title of protocols in order of display are in road signs of detours (right). Roman numerals I, II, and III correspond to the section of additional tips (1, 7, and 9 respectively).

Figure 2

Three step immunofluorescence method: The first antibody (orange) is binding to an epitope (Ag). In turn, primary antibody is recognized by a secondary antiserum (blue). This antibody is coupled to biotin (B), and this molecule can be observed under microscope by the binding of streptavidin-conjugated fluorochrome complex.

Figure 3

BS³ (bis(sulfosuccinimidyl)suberate) crosslinking: Linear chemical structure (upper); crosslinking reaction: the amino groups of proteins react with the N-hydroxysuccinimide (NHS) residues (middle); forming an amide bond (lower). Protein was adapted from “Icon Pack”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates. Accessed on 23 November 2021. Abbreviation: BS³ (Sulfo-DSS).

Figure 4

Key components of immuno-electrophoresis method: (A) 1% agarose gel. Loading wells must be positioned at the center to facilitate the detection of net charges of the antibodies and reagents; (B) suggested microtubes to adapt a tissue–antibody column; (C) place fresh 1% agarose (50–60 °C) in the bottom of selection tube, wait for polymerization, place the tissue in the proper orientation (layer of interest must go face up) over-polymerize agarose and fill gently with agarose and Triton X-100 in TGB as indicated in Step 1 of immunoelectrophoresis, then cut the plastic base of the tube and start to construct the column as indicated in the protocol; (D) column layers (1), tissue layer (2), antibody or reagent fluorochrome-conjugated layer (3), cover layer. Green arrow indicates the direction of migration. The green arrow indicates the direction of migration, which will depend on the net charge determined.

Figure 5

(A) Scheme of the effect of the antibody signal enhancer (ASE) over a eukaryotic cell. ASE solution components are indicated. Together, nonionic detergents Triton X-100 and Tween 20 poke holes in the membrane (enhanced permeabilization), and then antibodies and the rest of the ASE components (glycine and hydrogen peroxide) can get inside the cell. Frequently, some cell proteins are over-fixed, typically by paraformaldehyde or other aldehyde-fixing agents, and this excess is chelated by glycine molecule amino groups, favoring the binding of the antibody to the corresponding epitopes. Furthermore, hydrogen peroxide at micromolar concentrations successfully reduces autofluorescence without the quenching of the specific fluorescence or enzymatic signal, enhancing the specific signals, and reducing background noise from autofluorescence proteins or endogenous peroxidase from different cell compartments, such as membranes (e.g., GPCRs), cytoplasms (e.g., collagen complex) or organelles (e.g., lysosome, peroxisome). ASE can also enhance the signal of antibody-bound cytoplasmic proteins in transit from the nucleus or endoplasmic reticulum into their specific cell compartments previously permeabilized by both detergents (Triton X-100 and Tween 20); (B) Representative micrographs of Bicep femoris of male Wistar rat neuromuscular junction displaying immunofluorescence labeling (IF) and a staining reagent (Sr). In green is the postsynaptic nicotinic acetylcholine receptor (stained with α-bungarotoxin-Alexa Fluor^® 488 conjugate). In red label, given by secondary antibody Alexa Fluor^® 546 conjugate, is a representative immunofluorescence for S100 β-AF546 in perisynaptic Schwann cells. The immunostaining of Bicep femoris was performed under two different conditions using ASE method (upper), and using 2% BSA/0.2% Triton X-100 blocking solution as antibody incubation buffer (ASE free). The increase of signal using the ASE treatment was 3.339 for immunofluorescence, whereas 1.857 was the increase for fluorochrome-conjugated staining reagent. Intensity values were obtained using ZEN 2.6 blue edition Zeiss software (See Supplementary Materials).

Figure 6

Schematic representation of green fluorescent protein (GFP)-fluorescence emission in a eukaryotic cell: In situ environment, the cellular spread of GFP-emitting fluorescence (left). GFP under chemical fixation with paraformaldehyde (PFA); in some PFA–GFP complexes, a conformational change of GFP is induced, and as consequence GFP remains non-fluorescent (right). Based on Pereira et al. [47].

Figure 7

Super glue method for optimal preservation of retina: Schematic representation of mouse ocular globe and its anatomical structures (upper). Super glue attached from tip of the pipette to the mouse cornea (left), microtube Eppendorf containing super glue until limits of sclera; next, submerge immediately (1–2 s) into PBS-filled Eppendorf and remove excess of PBS. Poke micro-hole in the edge of the cornea, extract complex tip-cornea and lens (right), and proceed with fixation step. Figure based on Yang et al. [32].

Figure 8

Ultracold fixation method: (A) components for cryofixation: bottom to top, source of liquid (L) nitrogen and holder (gray) to put two methanol containers (1 and 2 in cyan); (B) tweezers previously submerged in silicone grease holding sapphire coverslips 15° from vertical, plunged in ultracold methanol for a few seconds, transferred to another box with ultracold methanol, unfreeze and monitored at proper temperature to find plasmatic membrane, extracellular proteins, or proteins in the cytoplasm; (C) honeycomb pattern of nucleus given by ice crystals and revealed by the DAPI stain. A and B based on Hagedorn et al. [28].

Figure 9

Timeline of antibody penetration: Left, standard immunofluorescence method is not adequate for antibodies to reach deep structures such as the nucleolus and its components, namely the granular, fibrillar center, and dense fibrillar (represented by circles within the nucleolus). Under conventional immunolabeling method range an antibody penetration between 8–9 µm has been reported [43]. Right, deep structures are reached by antibodies after trypsin treatment.

Figure 10

Unmasking antigenic sites by methods to induce epitopes retrieval (IER): Antigenic site (red) is not reachable for antibody binding (left) before unmasking treatments. After heat (HIER) or proteolytic (PIER) treatment antigenic sites are available for antibody binding (right).