key: cord-0759859-27lgp2u0 authors: Jorgensen, Marda; Joseph, Paul; Posgai, Amanda L.; Vander Heide, Richard S.; Kusmartseva, Irina; Atkinson, Mark A. title: ACE2 Chromogenic Immunostaining Protocol Optimized for Formalin-fixed Paraffin Embedded Human Tissue Sections date: 2021-07-21 journal: STAR Protoc DOI: 10.1016/j.xpro.2021.100696 sha: 9b5023540328d4b5f0f72d3141dc59f4d7575a25 doc_id: 759859 cord_uid: 27lgp2u0 Angiotensin-converting enzyme 2 (ACE2) is a key cellular entry factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, identifying cell types that express ACE2 is important for understanding the pathophysiology of coronavirus disease 2019 (COVID-19). We performed extensive testing of multiple primary antibodies across various human tissue types. Here we describe an optimized protocol for immunostaining of ACE2 in formalin fixed paraffin embedded human (FFPE) pancreas, small intestine and kidney tissue sections obtained from organ donors and autopsies. Angiotensin-converting enzyme 2 (ACE2) is a key cellular entry factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, identifying cell types that express ACE2 is important for understanding the pathophysiology of coronavirus disease 2019 . We performed extensive testing of multiple primary antibodies across various human tissue types. Here we describe an optimized protocol for immunostaining of ACE2 in formalin fixed paraffin embedded human (FFPE) pancreas, small intestine and kidney tissue sections obtained from organ donors and autopsies. For complete details on the use and execution of this protocol, please refer to (Kusmartseva et al., 2020) . The protocol below describes the specific steps for staining ACE2 in formalin-fixed paraffin embedded (FFPE) human pancreas, small intestine, and kidney tissue obtained from organ donors and at autopsies. However, with appropriate optimization, this protocol could potentially be applied to many other human tissue types (Hamming et al., 2004) . Human tissue samples 1. Process human tissues to formalin fixed paraffin embedded (FFPE) blocks using standard methods (Sadeghipour and Babaheidarian, 2019) . Note: Human tissue samples in the original study using this protocol (Kusmartseva et al., 2020) were collected by the Network for Pancreatic Organ donors with Diabetes (nPOD) (https://www.jdrfnpod.org/) program, as approved by the University of Florida Institutional Review Board, and at the University Medical Center New Orleans (New Orleans, LA), as approved by the Louisiana State University Institutional Review Board; both in accordance with federal guidelines involving informed consent from each individual's legal representative. The nPOD Standard Operating Procedure (SOP) that outlines protocol for histological preparation of nPOD samples could be found here. Assessment of tissue quality in postmortem samples 2. Using a microtome, cut 4m sections from FFPE tissue blocks. 3. Prior to selecting and staining tissue sections from an organ donor or autopsy case, stain a consecutive serial section with hematoxylin and eosin (H&E) according to manufacturer's instructions (Abcam). Note: ThermoScientific Micron HM 325 microtome was used in this protocol. Other commercially available microtomes are also suitable. Note: 4µm section thickness is recommended for the majority of tissue types. Optional: Other commercially available H&E kits are also suitable. 4. Visually assess tissue quality. a. If the tissue shows sufficient tissue quality (i.e., preserved tissue architecture with clearly identifiable tissue-specific structures; example of pancreatic tissue, Fig. 1A) , proceed with ACE2 staining. b. In contrast, if the tissue shows significant (>30%) signs of autolysis and advanced degradation (example of pancreatic tissue, Fig. 1B) do not attempt ACE2 staining as false positive signaling may occur. Timing: 1 hour J o u r n a l P r e -p r o o f 5. Preheat a dry oven to 55 o C. 6. Check that all reagents in the deparaffinization and rehydration containers are at a sufficient level to cover the entire surface of the slides. 7. Add tap water to a bench top staining tray just enough to create a humidified environment for performing the slide staining step. Step-by-Step Method Details Timing: 2 hours Paraffin is removed followed by hydrogen peroxide quenching to avoid detection of endogenous peroxidase activity, and tissue is rehydrated through incubation in graded ethanol solutions. Note: Fresh xylenes and graded ethanol solutions should be prepared daily for use in this protocol. For large numbers of stained slides, we recommend replacing these solutions after processing every 50 slides. Note: Deparaffinization and rehydration steps should be carried on in a chemical hood to minimize exposure to harmful chemicals. 1. Remove paraffin from FFPE sections. a. Melt paraffin to drive off any residual water trapped behind sections. i. Place slides in a xylene compatible, non-metal slide rack. ii. Bake the rack of slides in a 55 o C dry oven for one hour. b. Dissolve surrounding and infiltrating paraffin from the sections. Perform all steps at room temperature (20-22 o C). i. Place rack of slides in xylene for 5 minutes. ii. Transfer rack of slides to fresh xylene, incubate for 5 minutes. iii. Transfer slides to fresh xylene, incubate for 5 minutes. c. Clear the xylene from slides. Perform all steps at room temperature (20-22 o C). i. Place rack of slides in 100% ethanol for 5 minutes. ii. Transfer rack of slides to fresh 100% ethanol, and incubate for 5 minutes. Note: It is recommended that two slides be prepared for each case and tissue so that rabbit IgG isotype control can be stained in parallel with the ACE2 stained slide. To confirm the antibody specificity, preincubation of the ACE2 antibody with the synthetic peptide to which the antibody was raised could be utilized as an additional staining control. 2. Block endogenous peroxidases. a. Place rack of slides in freshly prepared 3% hydrogen peroxide diluted in methanol, and incubate for 10 minutes at room temperature (20-22 o C). Optional: Peroxidase blocking can alternatively be performed after the antigen retrieval steps using a commercially available hydrogen peroxide blocking reagent following the manufacturer's instructions. Pause Point: The assay could be paused at step 1 b (i) provided the incubation time does not exceed 72 hours. Note: Used xylene and ethanol solutions must be disposed of according to federal, state and organization laws. Critical: From this point on, do not allow slides to become dry as background will occur in the staining. Timing: 30 -40 minutes Heat retrieval is used to reverse fixation crosslinking and enable antibody recognition of the antigen. 4. Prepare retrieval chamber. a. Prepare 1X citrate buffer by diluting 1 part of10X citrate buffer stock solution with 9 parts deionized water. b. Transfer slides from rack to a plastic coplin jar filled with 1X citrate buffer. Note: Slides may be placed back-to-back in the coplin jar to increase capacity. c. Adjust the citrate solution level to cover the top of the slides. d. Place coplin jar into a 400mL Pyrex beaker and fill beaker with tap water up to one cm from cap edge. Note: Leave lid to coplin jar slightly loose to allow steam to escape. 5. Retrieve tissue sections. a. Program a microwave oven at 600 watts for 7 minutes. b. Place coplin/water jacket assembly in center of the microwave and press start. c. When microwave cycle ends, remove beaker from microwave and tighten lid. d. Allow coplin/water jacket assembly to cool on the bench top for 20 minutes. e. Incubate slides 1 minute in DI water to rinse off buffer. f. Using PAP pen, make a square or circle hydrophobic barrier around the tissue section to keep staining reagent localized to the tissue and minimize reagent waste. g. Place slides in prepared humid staining chamber and cover section surface in wash buffer. Optional: Use of humid staining chamber is strongly recommended. However, alternatively, use of PAP pen alone should create humid environment and keep tissue sections from drying provided that the incubation time with each staining reagent does not exceed the recommended duration. Optional: Borg Decloaker RTU (Biocare Medical, LLC) can be used as an alternative antigen retrieval solution, following manufacturer's instructions. Optional: Heat retrieval methods involving a different heating device, such as a water bath, have been successfully used in this assay. For the water bath epitope retrieval, we recommend the following setting: temperature 97 o C; time 30 minutes. The antigen retrieval solution should be pre-warmed to 97 o C before adding it to coplin jar with slides. The temperature in water bath should be closely monitored to ensure correct heat. Optional: It is possible to adapt this method to use a preferred retrieval reagent provided titer is tested and adjusted. Note: Ensure that serum matches the species that the secondary antibody is produced in. Pause Point: The assay can be paused for up to 2 hours provided the goat serum solution level is adequate for the tissue to remain completely wet. Timing: 60 minutes -overnight (8 to 12 hours) Diluted primary antibody solution is placed on top of tissue sections to allow recognition of the ACE2 antigen. 8. Perform primary antibody incubation. a. Dilute rabbit monoclonal ACE2 primary antibody at 1:150 in antibody diluent reagent. b. Wipe serum from the surface of the slide with a folded Kimwipe, avoiding contact with the tissue. c. Cover tissue section-containing area of slide with ACE2 antibody solution. d. Incubate for 1 hour. Optional: In a separate tube, dilute rabbit IgG isotype control at a final concentration to match that of the ACE2 antibody. Cover tissue section-containing area of an additional slide with isotype control antibody solution. Note: Concentration of antibody used is dictated by retrieval method. Alterations to the retrieval conditions will necessitate adjustment of the primary antibody titer. The assay can be paused by placing the humidity chamber at 4 o C and incubating overnight (8-12 hours). c. Cover tissue section in prepared ABC reagent and incubate for 30 minutes. d. Wash slides in wash buffer for 5 minutes. Pause Point: The slides can be held up to 10 minutes in buffer prior to performing chromogenic development. Timing: 30 -40 min. Chromogenic detection of positive signal is achieved using DAB, and the section is counterstained with hematoxylin to enable visualization of unstained structures. Note: DAB should be prepared immediately prior to use in order to avoid reagent precipitation. 11. Detect positive signal with ImmPACT DAB. a. Prepare working solution of DAB following kit instructions (combine one drop of DAB stock with one mL of DBA diluent). b. Wipe buffer from the surface of one slide with a folded Kimwipe, avoiding contact with the tissue. c. Cover surface of section in DAB solution and start a count-up timer. d. During DAB incubation, monitor brown color development under a brightfield microscope, typically 25-30 seconds. e. When the signal reaches the desired intensity, immediately stop the reaction by submerging the slide in water and rinsing with mild agitation. f. Place completed slide in a slide rack. g. Repeat development (steps b-f) for all remaining slides. 14. Clear the slides by placing them twice in xylene for 5 minutes. 16. Let slides dry for at least 1 hour. 17. Scan stained slides and view digitized images using slide scanner system such as Aperio CS2. Note: Use fresh hematoxylin solution after every batch of 50 slides. Used hematoxylin solution must be disposed of according to federal, state and organization laws. Optional: Alternatively, any xylene compatible permanent mounting media could be used to mount glass coverslips to the slides. Optional: Alternatively, staining results can be observed using a brightfield microscope. The described method consistently produces staining of specific sites in each organ tissue tested (Fig 2) . The enterocytes of the duodenum display strong ACE2 staining restricted to the brush borders of these cells ( Fig. 2A,B) . In the kidney (Fig. 2C,D) , intense, prominent ACE2 staining is observed in the apical brush borders of the proximal tubular cells. In contrast, the cytoplasm of these cells and endothelial cells of the vessels produce a weaker signal. Kidney glomeruli are negative for ACE2. In the pancreas, microvasculature endothelium in the islets and acinar tissue and some pancreatic ducts show distinct ACE2 positivity with tissue background devoid of signal (Fig. 2E,G) . The vast majority of larger pancreatic vessels are ACE2 negative. (Fig. 2E,H) . The ACE2 staining pattern in autopsy pancreatic tissue samples from patients with COVID-19 closely resembles the pattern of ACE2 expression observed in the pancreas of individuals without a history of SARS-CoV-2 infection (Fig. 3) . Staining results are heavily affected by the condition of the material tested. Adequate formalin fixation of non-degraded tissue is required. Problem 1: Tissue lifts from slide. Potential Solution: Use fresh sections mounted on plus charged slides. Problem 2: Staining of tissue fails in a cell type that is known to be positive. Potential Solution: ACE2 is highly expressed in microvasculature. If a positive control tissue fails, the assay should be repeated to confirm that all steps were included in stated order. Check DAB chromogen and kit component expiration dates. Problem 3: Staining of tissue is weak. Potential Solution: Increase chromogen incubation time while observing reaction development with a brightfield microscope. Problem 4: Poor signal to background discrimination. Confirm the tissue is staining appropriately by performing a primary antibody titration experiment. Repeat the experiment staining one slide each with 1:75, 1:150 and 1:300 primary antibody dilutions. Results should be in keeping with the serial dilution showing 1:75>1:150>1:300. Problem 5: Failure to find intense signal within autopsy tissue samples stained with the serially diluted primary antibody. Potential Solution: Confirm tissue quality by staining a consecutive tissue sections for H&E. If tissue shows less than 30% autolysis and degradation, repeat a primary antibody titration experiment by staining one slide each with wider range (1:20 to 1:600) of antibody dilutions. Do not attempt ACE2 staining if significant (>30%) autolysis and advanced degradation observed as false positive signaling may occur. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis Expression of SARS-CoV-2 Entry Factors in the Pancreas of Normal Organ Donors and Individuals with COVID-19 Making Formalin-Fixed, Paraffin Embedded Blocks We thank the families of the organ donors and autopsy patients for the gift of tissues.This research was performed with the support of the National Institutes of Health (R01 DK123292 to MAA) and the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF The authors declare no competing interests.J o u r n a l P r e -p r o o f Timing: 80 -85 minutes Biotinylated secondary antibody and avidin-biotin-horseradish peroxidase reagent are sequentially applied to the sections to recognize and amplify the antibody/antigen complex.Note: ABC reagent must be made according to manufacturer instructions prior to beginning the secondary antibody incubation so that the ABC completes a required 30-minute incubation step. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mark Atkinson (atkinson@pathology.ufl.edu). This study did not generate new unique reagents. This study did not generate/analyze datasets or code.