HIER buffer

Transfer the deparaffmized, rehydrated, and methanolic-peroxide blocked (optional) slides (see Notes 2-6) into plastic Coplin jars or containers filled with HIER buffer (see Note 7). (See Chapter 12 for preparation of tissue sections.)... 

Dako TRS, Dako Corporation, 6392 Via Real, Carpinteria, CA 93013 HIER buffer, Ventana Medical Systems, Tucson, AZ... 

Table 2 Guidelines for the use of HIER buffers and heading devices... 

Place the Coplin jars or plastic containers in the center of the microwave s turntable, cover containers with loose-fitting lids or screw caps, and heat at maximum power (700-1,200 W). The time of irradiation depends upon the power setting of the microwave, the type of container used, and the volume of buffer (tee Table 2). The solution should boil for 3-5 min. A large capacity microwaveable plastic container filled with 600-800 mL of HIER buffer may require a -30-45 min continuous heating cycle (13). 

Place the dewaxed/deplasticized and rehydrated sections (see Notes 7 and 8) in an MWPC filled with 1,500 mL HIER buffer (see Tables 1 and 2 and Note 18). 

Release the pressure carefully and cool the sections immediately in post-HIER blocking buffer (see Subheading 2, step 10) for 15-20 min before immunostaining (see Note 10). Alternatively, the slides can be allowed to cool down in the open pressure cooker (approx. 20-30 min) and then transferred into the post-HIER buffer (see Notes 5, 11, and 12). 

Preheat 3 L of HIER buffer (see Tables 1 and 2) to a boiling point in a stainless steel 5.5 L capacity pressure cooker (Prestige) without sealing the lid, using an electric hot plate as a heat source. Alternatively, to conserve buffer, the pressure cooker may be filled with distilled water, and a small loosely covered container filled with the selected buffer may be placed into the pressure cooker to hold the slides (see Note 18). 

Immerse the Coplin jar(s) into the water-bath and preheat HIER buffer (see Table 1) to the required temperature (95-99°C). 

Immerse the dewaxed/deplasticised and rehydrated slides into the preheated HIER buffer and cover the Coplin jar with a loose-fitting lid to avoid evaporation (see Notes 7 and 8). 

In most professional microwave ovens, power is regulated according to the set temperature, which is measured inside the HIER buffer. Some of these are complete histological microwave workstations and incorporate both large scale, rapid tissue processing under vacuum as well as HIER applications. Such instruments include, e.g. the Hostos Pro from Milestone Sri. (Sorisole, Italy) and the Meditest 800-3 series from Meditest Kft. (Budapest, Hungary). 

To reduce the use of costly commercial retrieval buffers (e.g. TRS) a small plastic container filled with the appropriate HIER buffer may be immersed within distilled water filling the MWPC chamber. 

Standardization of the conditions of HIER procedures is prudent. The power setting of the MWO, the number of the slides, the composition and volume of the retrieval medium, and the duration of the treatment are all interrelated. Always use standard power settings, either at a maximum or at limited power and titrate the procedure by adjusting the duration of retrieval or the buffer conditions. We would recommend using one or, at most, two HIER buffers for general applications and other buffer(s) only when absolutely necessary. 

There are several variations of HIER. Many laboratories have attempted to improve the original method by altering the buffer solutions as well as the source and mode of heating. Currently, the most popular HIER technologies use stainless steel or plastic pressure cookers, microwave ovens, or autoclaves as the heat source and low-molarity buffers with acidic or alkaline pR (6,7,9-12). 

The efficacy of the antigen retrieval procedure also depends on the treatment duration and the cooling conditions (see Subheadings 3.1.-3.5.), The power of the heating device and the buffer volume are among the major factors that determine the duration of a particular HIER protocol. 

Post-HIER immunostaining buffer 0.05 M Tris-buffered saline (TBS, pH 7.6) containing 1-2% fetal calf serum (FCS) or normal goat serum. 

Transfer slides to post-HIER immunostaining buffer, and continue with the immunohistochemical staining procedure (see Notes 10 and 12-16). 

Place the deparaffinized, rehydrated slides in metal or heat-resistant plastic slide racks and immerse in an autoclaveable incubation container filled with 250 mL HIER retrieval buffer (see Notes 2-7). Cover the container with a lid to avoid evaporation (see Note 10). 

As soon as the autoclave can be opened, the slides should be rinsed in post-HIER immunostaining buffer and used immediately in the immunostaining procedure (see Notes 11-16). 

The composition and the pH of retrieval buffers are crucial for optimal retrieval. Dako offers the following retrieval buffers for HIER. 

Despite some successes with the above pretreatments, the development of wet heat-induced epitope retrieval (HIER) procedures, which involves heating the fixed tissue sections in dilute metal-salt or buffer solutions at or above 100°C, for several minutes to 1/2 h, was the critical breakthrough in paraffin section immunohistochemistry (2, 7-9). Today, there are many variations of the original HIER technique. These differ primarily in the recommended buffer solutions and/or the source or mode of heating, but the basic formula of wet heat treatment over a fixed time period is similar. The most popular HIER technologies use microwave ovens, stainless steel or plastic pressure cookers, autoclaves, vegetable steamers or water-baths as the heat sources and low molarity buffers with acidic or alkaline pH (8,9,11-14). 

Experimental data suggest that the amount of heat and the duration of heating followed by the pH and chemical composition of the retrieval buffers are the most important factors for efficacy of HIER (7, 8, 15-18). Citrate buffer (sodium citrate-citric acid) at pH 6.0 is a very popular retrieval medium and has been used at molarities between 0.01 and 0.1 M (7, 8). Detergents (e.g., 0.1% Tween 20) added to the standard citrate buffer may improve the performance (14), but more prominent tissue deterioration and foaming may also be experienced. Other widely used antigen retrieval buffers are Tris-HCl at various concentrations (0.1-0.5 M) at... 

Common antigen retrieval buffers grouped according to their HIER efficiency and aggressiveness on tissue morphology... 

Incubate the slides in the post-HIER blocking buffer for 15-20 min (see Notes 10-12) then proceed with the immu-nostaining (see Notes 13-17). 

Since the achievable pressure and temperature in commercial pressure cookers vary among different models, the optimum duration of HIER must be determined for each instrument. Stainless steel pressure cookers usually allow heating up to 120°C and are more durable than those made of aluminum alloys which can be damaged by acidic or alkaline buffer solutions. Timer-controlled electric pressure cookers made of stainless steel are available at low price, e.g. Farberware Programmable Pressure Cooker, 4.5 L, and allow for easy standardization of HIER duration. 

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