TITLE: Cyclic Heat Induced Epitope Retrieval (CHIER)
AUTHORS: Victor Dieriks

[DESCRIPTION]
Immunolabeling is a cornerstone technique in molecular biology and pathology, providing critical insights into protein localisation and function within biological tissues. However, one of the persistent challenges in immunolabeling is the effective unmasking of epitopes, which are often concealed in formalin-fixed, paraffin-embedded (FFPE) tissue samples. While effective to an extent, traditional antigen retrieval methods have limitations in consistency, epitope preservation, and overall labelling efficiency. We introduce a novel antigen retrieval protocol, Cyclic Heat Induced Epitope Retrieval (CHIER) to address these issues.
 
The development of CHIER is particularly crucial for studies involving proteins that are notoriously difficult to label due to dense cross-linking or subtle epitope masking. By improving the efficiency of antigen retrieval, CHIER enhances the sensitivity and specificity of antibody binding in immunohistochemical (IHC) and immunofluorescence (IF) assays. This results in more robust staining and can facilitate the detection of densely aggregated proteins, which is essential for accurate protein localisation and quantification studies.
 
Significantly, CHIER demonstrated a remarkable ability to increase the detection of the protein of interest (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin subfamily c member 2, SMARCC2), with results showing a 3x increase in detection compared to traditional methods. This enhancement in detection efficiency makes CHIER a valuable tool for researchers in diverse fields, including neurobiology, cancer research, and tissue pathology, where precise antigen detection is paramount. Introducing CHIER into these fields is expected to significantly advance our understanding of complex protein dynamics and interactions in various biological and disease contexts.
 

[GUIDELINES]
Heat block reaches 70° Celcius and can cause burns when skin comes into contact with the hot surface.

[STEPS]
SECTION: Paraffin-embedded formalin-fixed brain tissue
1. Human paraffin-embedded formalin-fixed brain tissue was utilised for this project. All brain tissues were washed using an ethanol (EtOH) series at Room temperature :
20 min 70% EtOH
20 min 80% EtOH
20 min 2 x 95% EtOH 
30 min 2 x 100 % xylene 

Once washed and dehydrated, the tissue was embedded in molten paraffin wax for 3 x 25 min cycles. 
  

SECTION: Dewaxing
4. 
Immediately following hotplate incubation, sections were submerged in 
30 min 2 x 100% xylene baths to remove paraffin wax. 
Subsequently, tissue sections were rehydrated by submerging slides in a series of alcohol baths 
15 min 2 x 100% EtOH 
5 min 95% EtOH 
5 min 80% EtOH 
5 min 75% EtOH 
5 min 3 x distilled H2O (dH2O) to wash of any remaining EtOH. 
  

SECTION: Cutting parafin sections
2. Paraffin blocks were cut into 7 μm thick sections using a rotatory microtome (Leica Biosystem Histcore BIOCUT R) and placed into a 37 °C water bath (Leica Biosystems, Histcore & H1210). Sections were individually mounted onto charged Uber frost Printer PLUS 3000 microscope slides (Intrumentec) and placed on a slide dryer (Leica Biosystems Histcore) for 5 minutes, followed by air-drying at room temperature for a minimum of 4320 min. 

SECTION: Cyclic Heat Induced Epitope Retrieval (CHIER) Hotplate
3.2. Immediately following the heat-cooling cycle, slides were placed on a 60 °C hotplate (Leica HI1220) for 30 min.  
 

SECTION: Cyclic Heat Induced Epitope Retrieval (CHIER) Hotplate
3.1. Mounted slides with Formalin-fixed paraffin-embedded tissue sections were placed, on a 70 °C hotplate (Leica HI1220) for 5 min followed by 5 min at room temperature. 
This heat-cooling cycle was repeated six times.

 

SECTION: Cyclic Heat Induced Epitope Retrieval (CHIER) Hotplate
3. Mounted slides with Formalin-fixed paraffin-embedded tissue sections were placed tissue side up 

SECTION: Antigen retrieval in pressure cooker
5. The tissue on the slides is now ready for subsequent steps. Continue with antigen retrieval as normal, followed by blocking and subsequent antibody incubation.