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.
METHOD SUMMARY
An economical method of heat-mediated antigen retrieval using a low-cost, readily available Instant Pot® is described. This approach is easily controlled, safe and can be reliably used by individual research labs.
Tweetable abstract
“Cooking” with Instant Pot®: an easily controlled, safe, inexpensive and flexible method for performing heat-induced antigen retrieval for immunohistochemistry.
The current work describes and validates an inexpensive method to perform heat-induced antigen retrieval for immunohistochemistry (IHC) using an Instant Pot®. This approach makes it easier and less expensive for research labs to perform IHC on formalin-fixed paraffin-embedded tissues. Here, the feasibility of the method is demonstrated using several different monoclonal antibodies. IHC is well-established in human studies. It is a mainstay in clinical pathology labs and is the gold standard for the diagnosis of many diseases. However, the use of formalin-fixed mouse tissues has lagged behind for several reasons. One significant barrier has been the antigen-retrieval step that is needed for the detection of many antigens. While there are many procedures described for heat-induced antigen retrieval, they have significant limitations that make their use by individual research labs challenging. For example, high-throughput procedures that use specialized and relatively expensive equipment are not suitable for research labs that only sporadically perform IHC. Other approaches have used pressure cookers, hot water baths or microwave ovens for antigen retrieval. However, many of these less expensive approaches can result in the loss of valuable samples, present challenges in achieving a consistent temperature or have additional safety concerns [1–4]. Here, an alternative to other antigen-retrieval methods is presented that is easy, safe, reproducible and economical, making it accessible to individual research labs and even undergraduate lab courses.
An alternative antigen retrieval method that is easily performed using an inexpensive Instant Pot was developed. The method presented here utilizes an Instant Pot Ultra, purchased for approximately $130 from Amazon (WA, USA). This 3 qt. mini Instant Pot® can easily hold three glass Coplin staining jars, each of which holds multiple slides (Figure 1A). The Instant Pot temperature can be easily set and modified using the control panel (Figure 1A). After setting the Instant Pot Ultra to 97°C using the “ultra” setting, the temperature of both the water bath and the antigen-retrieval buffer inside the Coplin jars were measured after 5, 10, 15, 25 and 35 min. As shown in Figure 1B, the Instant Pot quickly heated up and was within 3°C of the set temperature at 25 min. Based on these results, Coplin jar slide holders with antigen-retrieval buffer (either Tris-EDTA buffer, pH 9.0, Abcam [Cambridge, UK] ab93684 or citrate buffer, pH 6.0, Abcam ab93678) were prewarmed in the Instant Pot for 25 min before slides were added for a 25-min antigen-retrieval incubation. To confirm appropriate temperature maintenance during the 25-min antigen-retrieval incubation, the temperature of the retrieval buffer in the Coplin jars was measured after the incubation and was 95°C, or within 2°C of the set temperature. The IHC antigen-retrieval procedure was thus set as a 25-min prewarm incubation of buffer-filled Coplin jars in the Instant Pot prior to a 25-min incubation of the sample slides for antigen retrieval.
To illustrate the effectiveness and necessity of antigen retrieval by the Instant Pot method, an anti-CD8 antibody was tested on formalin-fixed paraffin-embedded sections of the transplantable Colon 38 (C38) tumor cell line grown in syngeneic C57BL/6 mice. This carcinogen-induced tumor cell line is known to contain many CD8 cells [5]. Slides were deparaffinized employing a standard series of xylene and ethanol washes [1,4]. C38 sections that either underwent the Instant Pot antigen retrieval or received no antigen retrieval were then compared prior to staining with an anti-CD8 antibody. As can be seen in Figure 2A–C, the section treated with the Instant Pot antigen-retrieval method shows strong positive staining of CD8 cells. In contrast, the sample that received no antigen retrieval treatment showed very little staining, clearly illustrating the necessity of antigen retrieval. The specificity of the staining was further tested on formalin-fixed paraffin-embedded spleen sections by staining serial sections of a C57BL/6 spleen with anti-CD4 or anti-CD8 rabbit monoclonal antibodies or isotype control. As can be seen in Figure 3A–C, the antibody staining is specific, as the isotype control showed little staining, while both the anti-CD4 and the anti-CD8 antibodies gave staining patterns consistent with the known distribution of these T-cell populations in the spleen. Moreover, as is also demonstrated in Figure 2 for the anti-CD8 antibody, antigen retrieval is essential for successful staining with the anti-CD4 antibody (Figure 3D–G). These studies were extended to include a variety of different antibodies (Table 1). Using the Instant Pot retrieval method, clear staining was seen using several different antibodies on both normal tissues as well as tumor tissues, which often have high background staining due to necrosis and additional cellular debris. The Instant Pot antigen-retrieval method can be used with cell surface antigens including CD4, CD8, NCR1, CD38 and CD31, as well as with intracellular antigens such as the transcription factors EGR-2 and FoxP3. These antibodies gave the expected staining patterns (Figure 4A–F), similar to what others have found using other antigen-retrieval methods with these antibodies [6,7]. For example, EMT6 sections stained with anti-CD31 gave characteristic staining of endothelial cells lining vessels, and anti-F4/80 revealed that EMT6 is heavily infiltrated by myeloid/macrophage cells as expected [8,9]. Both anti-EGR-2 and FoxP3 demonstrate intense punctate staining of nuclei, as predicted for transcription factors (Figure 4B & F). Of note, the data in Figure 4 demonstrate the efficacy of the Instant Pot antigen-retrieval method for IHC using both rabbit and rat monoclonal antibodies. Interestingly, rabbit monoclonals are often generated against peptides. As a result, rabbit antibodies often do not require the native structure of the protein to bind and may be of higher affinity [10]. Thus, rabbit antibodies may be especially effective in IHC. Importantly, any antibodies that can be successfully used with heat-induced epitope retrieval would be expected work with the Instant Pot method. Nonetheless, like other heat-retrieval methods, there may be some antigen-antibody combinations that will not work with the Instant Pot method. However, an advantage of this method is that the temperature can be easily modified in the mini Instant Pot® Ultra. Thus, this method can be readily optimized for individual antibodies, if needed, broadening its utility. Coupled with this broad utility, the Instant Pot method's ease of use, safety and low cost offer many advantages for performing 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.
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.
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.
| Target antigen | Type of antigen/primary cell type or tissue | Species of antibody | Clone/company |
|---|---|---|---|
| CD4 | Cell surface, helper T cells | Rabbit mAb | EPR19514/Abcam, catalog # ab183685 |
| CD8 | Cell surface, cytotoxic T cells | Rabbit mAb | EPR21769/Abcam, catalog # ab217344 |
| NCR1 (also called NKp46, CD335) | Cell surface, NK cells | Rabbit mAb | EPR23097-35/Abcam, catalog # ab233558 |
| CD31 | Cell surface, vessels and some macrophages | Rabbit mAb | EPR17259/Abcam, catalog # ab225883 |
| IgG isotype (no target) | Negative control | Rabbit IgG mAb | EPR25A/Abcam, catalog # ab172730 |
| FoxP3 | Intracellular (nuclear transcription factor) Tregs | Rat mAb | FJK-16s/Invitrogen, catalog # AB_467576 |
| CD38 | Cell surface antigen on a variety of immune cells including B cells and subsets of macrophages | Rabbit mAb | EPR20179/Abcam, catalog # ab216343 |
| EGR-2 | Transcription factor on a variety of cells including M2-like macrophages and some T cells | Rabbit mAb | EPR23228-40/Abcam, catalog # ab245228 |
| F4/80 | Cell surface glycoprotein found on numerous murine macrophages | Rabbit mAb | SP115/Abcam, catalog # ab111101 |
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.
Author contributions
K Norville prepared the tissue and tumor samples used for immunohistochemistry and was involved in the conceptual framework of the paper. N Kearns performed the antigen retrieval and staining of the tissues and contributed intellectually to the experimental design. J Frelinger conceived the overall concept of the paper. All authors were involved in writing and editing the manuscript.
Acknowledgments
The authors would like to thank their colleagues for helpful suggestions after presenting this work in seminars and Aditi Murthy for advice on immunohistochemistry for FoxP3.
Financial & competing interests disclosure
K Norville was supported in part by The American Association of Immunologists through a Careers in Immunology Fellowship and by the National Institutes of Health Training Grant AI07285. The project was supported in part by 1R21CA184433. The authors also acknowledge generous support from Steven and Alison Krausz and FC Blodgett. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval for animal experimental investigations. The animal experiments to obtain tissue or tumor samples were performed in accordance with guidelines established by the National Institutes of Health and approved by the University Committee on Animal Resources.
Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
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