Instant Pot for antigen retrieval: a simple, safe and economical method for use in immunohistochemistry

Abstract

Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot^® for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovens or scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safe and inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.

Keywords: antigen retrieval; heat induced; histology; immunohistochemistry; monoclonal antibody; tumor.

Figure 1.. Setup of the mini Instant Pot^® Ultra for antigen retrieval.

(A) Mini Instant Pot^® Ultra, control panel and Coplin jar used for antigen retrieval. The image highlights the “ultra” setting used for antigen retrieval, the temperature display and the central control knob used to set desired parameters. (B) Both water bath (black bars) and antigen retrieval buffer (white bars) temperatures were measured at various time intervals to establish the warm-up time needed to achieve a buffer temperature of approximately 95°C using the ultra setting on heat only. Glass Coplin jars were filled with 50 ml of antigen retrieval buffer and placed in approximately 2 quarts of water inside the mini Instant Pot^® Ultra. The water bath and buffer temperature were determined at various time intervals with the mini Instant Pot^® Ultra set to 97°C. A 25-min warm-up duration was chosen based on this initial experiment. After a 25-min warm-up, sample slides were placed directly into the heated buffer in the Coplin jars and, after a 25-min run, the buffer temperature was approximately 95°C (grey bar). This protocol for antigen retrieval was used for all subsequent experiments. The formalin-fixed paraffin-embedded sections (5 micron) were processed using standard procedures. The sections from the samples were treated using the Instant Pot antigen-retrieval method and subsequently probed with various antibodies as described in the figure legends and listed in Table 1. Secondary antibodies were either goat antirabbit IgG (H+L) biotinylated RTU (VectorLabs, CA, USA) or goat antirat IgG mouse adsorbed (H+L) biotinylated (VectorLabs) and were used in accordance with the manufacturer's recommendations. The sections were subsequently stained with hematoxylin.

Figure 2.. Antigen retrieval is required for successful staining of CD8 cells in Colon 38 tumor tissues and can be done using Instant Pot retrieval method.

C38, a murine adenocarcinoma, was grown in syngeneic C57BL/6 mice. Tumors were harvested, formalin-fixed and paraffin-embedded, and 5-micron sections were used in immunohistochemistry experiments. Serial sections either received or did not receive the antigen-retrieval protocol described in Figure 1. (A) C38 tissue section that did not receive antigen retrieval was stained with a rabbit antimouse mAb to CD8α, a cell surface marker expressed on cytotoxic T cells. (B) C38 tissue that received antigen retrieval was stained with an antimouse mAb to CD8α. (C) C38 tissue that received antigen retrieval was stained with a rabbit IgG isotype control mAb. For all panels, scale bar = 20 μm. mAb: Monoclonal antibody.

Figure 3.. Spleen cell sections treated using Instant Pot antigen retrieval method and stained with anti-CD4 or anti-CD8 mAb show expected staining pattern.

Spleens were harvested from C57BL/6 mice, formalin-fixed and paraffin-embedded, and serial sections then underwent the IHC protocol with the Instant Pot antigen-retrieval method. Spleen sections were stained with (A) rabbit antimouse mAb to CD8α, (B) rabbit antimouse mAb to CD4, a cell surface molecule characteristic of helper T cells or (C) IgG Isotype control mAb. (D & E) Low and high magnification of sections of spleen stained with anti-CD4 mAb without antigen retrieval and (F & G) low and high magnification of sections stained with anti-CD4 mAb with antigen retrieval illustrating the efficacy and necessity of antigen retrieval for this mAb. IHC: Immunohistochemistry; mAb: Monoclonal antibody.

Figure 4.. Instant Pot antigen-retrieval process was validated for several antibodies and can be used for both normal and tumor tissues.

Sections of C57BL/6 spleen or EMT6 tumors from a BALB/c mouse were stained with different antibodies after Instant Pot antigen retrieval to demonstrate the wide applicability of the technique. (A) C57BL/6 spleen stained with a rabbit antimouse mAb to NCR1 to visualize natural killer cells. (B) C57BL/6 spleen stained with a rat antimouse mAb to FoxP3 to visualize regulatory T cells. (C) EMT6 tumor tissue stained with a rabbit antimouse mAb to CD31 to visualize endothelial cells and some immune cells such as macrophages. (D) EMT6 tumor tissue stained with a rabbit antimouse mAb to F4/80 to visualize macrophages. (E) EMT6 tumor tissue stained with a rabbit antimouse mAb to CD38 to visualize infiltrating immune cells including B cells and macrophages. (F) EMT6 tumor tissue stained with a rabbit antimouse mAb to EGR2, a transcription factor expressed by a variety of leukocytes including subsets of macrophage populations. For all panels, scale bar = 20 μm. mAb: Monoclonal antibody.