Identification of Cells with Markers of Cellular Senescence in Human Formalin-fixed, Paraffin-embedded Brain Tissue Sections

Summary

We describe an immunofluorescence procedure to detect markers of cellular senescence in human formalin-fixed, paraffin-embedded brain sections. In contrast to immunohistochemistry, immunofluorescence permits semi-quantitative measurements and multiple staining on the same slide, which allows detecting different cell types more rigorously and assessing the abundance of senescence markers more objectively.

Abstract

Cellular senescence is a terminal arrest in proliferation associated with aging and age-related diseases. Senescent cells are characterized in vitro by several morphological and biochemical markers, such as flattened morphology, Senescence-Associated beta-galactosidase activity, chromatin rearrangements, and expression of the cyclin-dependent kinase-inhibitors p21 and p16^INK4A.

Here we describe a procedure to detect p16^INK4A-positive cells by immunofluorescence on formalin-fixed, paraffin-embedded, human-brain specimens. Briefly, tissue slides are deparaffinated, rehydrated, and subjected to antigen-retrieval with standard immunohistochemistry procedures. The slides are then incubated with serum to block non-specific binding sites, and with primary antibodies against p16^INK4A and a specific cell type marker. Finally, the slides are stained with fluorescently-conjugated secondary antibodies and counterstained with DAPI. This technique has been used successfully to detect p16^INK4A positive astrocytes in human brains, but can be easily applied to other cell types, markers, and tissues, provided no cross-reactivity ensues between the antibodies.

Our technique allows for the identification of subpopulations of senescent cells more rigorously than immunohistochemistry and enables the user to measure the abundance of a senescence marker semi-quantitatively.

Introduction

Cellular senescence is a terminal arrest in cell proliferation ¹ in response to telomere attrition, DNA damage, oncogenic activation, and oxidative stress ^2,3 . Cells expressing markers of senescence have been found in vivo in skeletal muscle, lung and liver of old animals ⁴ and recently we have demonstrated the presence of senescent astrocytes in human brain ⁵. Senescent cells contribute to the aging process and to the onset of age-related pathologies by secreting several pro-inflammatory cytokines ⁶, a phenomenon known as Senescence-Associated Secretory Phenotype (SASP) ⁷. At the cellular level, senescence is characterized by several distinctive markers, such as senescence-associated β-galactosidase activity ⁸, accumulation of heterochromatin protein 1 and of the histone variant macro H2A ^9,10 , secretion of interleukin 6 and matrix metalloproteinase 1 ^5,7 , and increased expression of the cyclin-dependent kinase-inhibitor p16^INK4A. Although these markers are not exclusive to senescent cells, expression of p16^INK4A is considered to be a robust indicator of cellular senescence in vivo ¹¹ and has been used successfully in human specimens ^5,11 . p16^INK4A-expressing cells appear to be a critical factor in age-related pathologies: p16^INK4A-positive astrocytes accumulate in human brains affected by Alzheimer's disease ⁵, and clearing p16^INK4A-expressing cells delays aging in rodents ¹².

We describe here a method for detecting cells expressing p16^INK4A in formalin-fixed, paraffin-embedded (FFPE), human-brain specimens by immunofluorescence ⁵. Our protocol offers several advantages in comparison to immunofluorescence on frozen sections or immunohistochemistry: i) it can be used on tissues from most pathology autopsy archives (we provide an example on frontal cortex autopsy-specimen archival-tissue), ii) it is suitable for multiple staining on the same tissue slide, thus allowing discrimination between cell types based on the expression of specific cell markers, iii) it can be coupled with regular immunohistochemistry and thus with staining protocols clinically approved for diagnostic use.

Protocol

Human Subjects Ethics Statement

This research was performed in compliance with and following approval by the Institutional Review Board at Drexel University College of Medicine (Protocol Number: 18172), and the Institutional Review Board at University of Pennsylvania (Protocol Number: 180600). All tissue samples were de-identified and no protected health information was made available to the researchers; therefore, this protocol was approved as exempt study and consent was waived.

1. Deparaffination and Rehydration of Tissue Sections

Note: Once the protocol has been started, ensure that the slides are never allowed to dry out because this could interfere with quality of the staining.

1. Deparaffinize tissue sections on glass slides by immersing slides into a glass dish filled with xylene for 5 min (CAUTION: Use only under a chemical fume hood, possible cancer hazard). Transfer slides to the next dish filled with fresh xylene and incubate for 5 min and repeat this step once more for a total of 3 incubations of 5 min each. Keep track of how often the xylene in each container was used and properly discard of xylene after 3 uses.

Note: Set up and turn on heat steamer as described in point 2.2

2. Transfer slides to the next dish filled with 100% alcohol (CAUTION: flammable) and wash for 5 min. Repeat once more with fresh 100% alcohol for a total of 2 X 5-minute washes.
3. Prepare 90% alcohol by diluting with deionized water (i.e. 90 ml of alcohol + 10 ml of deionized water). Incubate slides in 90% Reagent Alcohol for 5 min. Repeat once more with fresh 90% alcohol for a total of 2 X 5-minute washes.
4. Prepare 80% alcohol by diluting with deionized water. Incubate slides in 80% alcohol for 5 min. Repeat once more with fresh 80% alcohol for a total of 2 X 5-minute washes.
5. Prepare 70% alcohol by diluting with deionized water. Incubate slides in 70% Reagent Alcohol for 5 min. Repeat once more with fresh 70% alcohol for a total of 2 X 5-minute washes.
6. Incubate slides in deionized water for 5 min. Repeat once more with fresh deionized water for a total of 2 X 5-minute washes.

2. Antigen Retrieval

1. Transfer slides to a horizontal slide holder filled with 10 mM citrate buffer pH 6.0. Equilibrate slides in this buffer for 5 min. Prepare 10 mM citrate buffer pH 6.0 by diluting from 100 mM stock of citrate buffer. For 1L of 100 mM citrate buffer stock pH 6.0, dilute 21.01 g of citric acid monohydrate (formula weight 210.14 g/mol) in distilled deionized water, bring the pH to 6.0 with a solution of NaOH, and then adjust the volume to 1L.
2. Heat-steam slides in a covered dish filled with citrate buffer for 15-20 min depending on the degree of cross-linking of the tissue sample. Heat steamer (rice cooker) should be set-up and turned on during deparaffination steps by filling lower chamber approximately halfway with distilled water.
3. Remove covered dish with slides in citrate buffer from heat (CAUTION: dish will be hot) and place on lab bench top for at least 30 min.
4. Wash slides 2 X 5 min in 1X PBS 0.1% BSA solution. (For 1 L of 1X PBS-0.1%BSA solution, add 1 g of bovine serum albumin (BSA) to 1 L of phosphate-buffered saline (1X PBS) and mix well to dissolve BSA).

3. Blocking

1. Prepare a humidified chamber by placing filter paper in the bottom of a plastic slide box and moistening with deionized water.
2. Prepare blocking solution to a final concentration of 0.1% BSA (w/v) 0.25% Triton X-100 (v/v), 5% normal goat serum (v/v), and 5% normal donkey serum (v/v) in 1X PBS. The type of serum used in the blocking solution will depend on the species in which the secondary antibodies were raised (i.e. If using donkey anti mouse secondary antibody and goat anti rabbit secondary, block with donkey and goat serums simultaneously).
3. Remove slides one at a time from the PBS-BSA wash and wipe away excess liquid from the back and edges of the slide. Avoid making any contact with the tissue section.
4. Place slide in humidified chamber. Draw a hydrophobic border around the outside of the tissue section with a PAP pen. When circumscribing tissue area, ensure that area through which the line is drawn is completely dry; otherwise the border may be lost during immunostaining. Avoid putting too much pressure on the pen, which could result in excess solution being discharged onto the slide.
5. Immediately add enough blocking solution to cover the tissue. Use approximately 100-200 microliters per tissue section, depending on the surface area. Avoid contacting the tissue section with the pipette tip. Repeat for all slides.
6. Incubate slides with blocking solution for 90 min in the humidified chamber at room temperature.

4. Immunoflourescence Staining with Two Primary Antibodies Simultaneously

1. For immunostaining with more than one primary antibody simultaneously, (i.e. a senescence biomarker (mouse anti-p16^INK4A) and a cellular biomarker (rabbit anti-GFAP (glial fibrillary acidic protein) to visualize astrocytes), dilute primary antibodies to desired final concentration in antibody dilution buffer consisting of 1X PBS, 0.1% BSA (w/v), and 0.25% Triton X-100 (v/v) in the same 1.5 ml tube and mix well. See Discussion regarding selection of primary antibodies.
2. Blot off blocking solution by pressing the edge of the slide against filter paper.
3. Apply the solution of primary antibodies to each slide. Use approximately 75 microliters per slide or enough volume to cover depending on tissue surface area.
4. Incubate in humidified chamber overnight (approximately 16 hr) at 4 degrees Celsius. Handle humidified chamber carefully to avoid displacing primary antibody solution from the tissue area.
5. Blot off primary antibody by touching the edge of the slide to filter paper. Wash slides in 1X PBS 0.1% BSA for total of 3 x 5-minute washes.
6. Prepare secondary antibodies (conjugated to different fluorochromes) by diluting to a final concentration of 1: 500 in antibody dilution buffer (see step 4.1). Incubate in the dark in a humidified chamber for 1 hr. The secondary antibodies are light-sensitive; therefore, this step and all subsequent steps should be protected from light.
7. Blot off secondary antibody solution by touching the edge of the slide to filter paper. Wash slides in 1X PBS 0.1% BSA for total of 3 x 5-minute washes.
8. Prepare DAPI working solution by diluting DAPI (50 μg/ml stock) 1:5000 with 1XPBS 0.1%BSA. Apply the DAPI working solution to the slides and incubate for 10 min in the dark.
9. Rinse slides abundantly with at least 3-4 washes with distilled deionized water.

5. Mounting Slides for Immunofluorescence Microscopy

1. Remove slides one at a time from the water wash and wipe away excess liquid from the back of the slide. Avoid making any contact with the tissue section.
2. Place a drop (approximately 10 microliters) of fluorescence mounting medium in the center of the tissue section. Avoid using too much mounting medium, otherwise the coverslip will move against the tissue section and it will be difficult to seal the edges.
3. Place a glass coverslip (24 x 50 mm) on edge of slide and slowly lower onto the tissue section to avoid generating bubbles.
4. Seal coverslips with clear nail polish by first putting a drop of nail polish at one corner of the slide with coverslip and dry undisturbed for approximately 5 min. Seal the remaining corners with nail polish and then seal the short and long edges of the coverslip. Allow nail polish to try before going to the microscope.
5. Store stained slides at 4 degrees Celsius and visualize within two weeks.

6. Image Acquisition and Analysis

Using a fluorescence microscope, capture images using the appropriate channels depending on the fluorochromes used.

OPTIONAL: Combined Immunohistochemistry and Immunofluorescence

Prior to immunofluorescence staining, immunohistochemistry can be performed in the same tissue section for an additional marker. Following antigen retrieval (Step 2), sections can be stained using standard immunohistochemical methods in the absence of a counterstain. Slides are visualized with the fluorescence microscope and captured in brightfield.

Results

We demonstrate a method for detecting a senescence biomarker within a specific cell type in situ in archived FFPE human brain tissue. Figure 1 shows representative images of p16^INK4A and GFAP staining in AD patient and an age-matched control subject. We demonstrate that p16^INK4A staining is diffusely localized throughout the nucleus, while GFAP staining is cytoplasmic. Nuclear p16^INK4A staining is considered positive when it colocalizes with DAPI and the signal inte...

Discussion

Double immunofluorescence labeling of human brain tissues is a well-established method in the study of neurodegenerative disease ^13-15 and methods for performing immunofluorescence on FFPE tissues have been described previously ¹⁶, thus we redirect the reader to them for a more detailed discussion of the technique, while we will focus on key concepts necessary for applying the technique successfully to detect senescent cells. Background signal and antibody specificity are the most critical ch...

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Xylene	Fisher Scientific	X3P-1GAL	histological grade, flammable
Reagent Alcohol	Fisher Scientific	A962F-1GAL	flammable liquid
Citric acid monohydrate	Sigma Aldrich	C7129-500G	eye irritant
Phosphate-buffered Saline	Mediatech	46-013-CM	10X
Bovine Serum Albumin	Gemini Bioproducts	700-101P
Triton X-100	Sigma Aldrich	T-9284	irritant
ImmEdge Pen (PAP pen)	Vector Laboratories	H-4000
Goat Serum	MP Biomedicals	191356
Donkey Serum	Millipore	S30-100ML
Mouse anti p16 antibody	Santa Cruz Biotechnology	sc-1661
Rabbit anti GFAP antibody	Millipore	AB5804
Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)	Life Technologies	A-11008
Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L)	Life Technologies	A31570
DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride)	Sigma Aldrich	32670-5MG-F
Vectashield Mounting Medium	Vector Laboratories	H-1000
Coverslips	Fisher Scientific	12-544-14	24x50 mm
Clear nail polish	Any supplier
Heat steamer	Any supplier		rice cooker
Olympus BX61 fluorescence microscope	Olympus
Hamamatsu ORCA-ER camera
Slide Book 4 Software	Intelligent Innovations, Inc	version 4.0.1.44