Immunofluorescence staining of kidney sections to monitor ischemia-induced damage

doi:10.1038/protex.2016.005

Method Article

Immunofluorescence staining of kidney sections to monitor ischemia-induced damage

https://doi.org/10.1038/protex.2016.005

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The proper cellular and subcellular localization of proteins is critical to organ performance under physiological and pathological conditions. Reliable procedures to monitor changes in this distribution are required in experimental medicine and for clinical diagnosis. The kidney is particularly relevant to human health, because its damage is associated with various different diseases. Here, we describe a protocol for immunofluorescent staining of kidney sections. The procedure is performed on two consecutive days; it takes less than 5 hours to complete. Major strengths of the protocol are the conservation of tissue morphology, low background and the possibility to assess multiple proteins at the same time. Our protocol was applied to evaluate the effects of ischemia-reperfusion injury on kidney cells. The methods described by us were used to reveal ischemia-induced changes in cell morphology and protein distribution.

Cell biology

Biological techniques

Computational biology and bioinformatics

microscopy

immunofluorescence

immunohistochemistry

ischemia

reperfusion

kidney injury

The function of proteins is frequently linked to their subcellular localization. Immunostaining, a common technique to detect cell type-specific gene expression, also monitors changes in protein distribution and abundance [1, 2]. In clinical nephrology immunostaining is still essentially limited to the detection of endogenous immunoglobulins and complement pathway proteins on frozen tissue sections. However, immunofluorescence techniques to characterize more architecturally preserved paraffin embedded tissue are increasingly used for research purposes and open the door to a deeper understanding of specific cellular and subcellular events.

Unlike chromogenic immunohistochemistry, fluorescence immunostaining of tissues provides high specificity and simplifies multiplexing for the simultaneous detection of several antigens. This multiplexing is particularly useful to analyze heterogeneous organs or tissues that contain different cell types which are identified with specific markers. An example of such a complex organ is the kidney. The cortex and medulla represent major layers of the kidney. Embedded in these layers are different parts of the nephron, including glomerulus, proximal tubule, ascending and descending limbs of Henle, distal tubule and collecting duct (reviewed in [3]). As a result of the spatial organization of the nephron, the kidney cortex and medulla contain different tubular cell types, which can be distinguished by marker proteins [4, 5]. Protein profiles and distribution vary in each tubular segment. For example, aquaporin-1, a water channel in the plasma membrane, is abundant in proximal tubules but absent in other tubular segments [6, 7].

Healthy cells of the proximal tubule are polarized; their apical and basolateral membrane domains differ in protein composition [1]. Under normal conditions, aquaporin-1 is present in high concentrations in the apical domain, but can also be detected in the basolateral domain [4]. Disease or aging often compromise the polarized membrane organization of kidney epithelial cells; this may cumulate in the improper distribution of proteins in the plasma membrane domains [1]. Such relocation is not limited to plasma membrane proteins, but affects other subcellular compartments as well. Ischemia-reperfusion injury (IRI) can result in acute kidney damage and ultimately kidney failure [8, 9]. As a consequence of IRI, the polarized organization of proximal tubule cells is impaired, and the separation of apical and basolateral membrane domains becomes compromised.

Reliable protocols are necessary to evaluate the cellular and subcellular changes in the kidney that determine renal function under health and disease conditions. Methods that identify specific kidney cell types and monitor a protein of interest will greatly enhance these analyses. To this end, we describe protocols that are compatible with multi-color fluorescent immunostaining of kidney sections. We used our procedures to assess the subcellular localization of the water channel aquaporin-1 [4, 6] and HuR, an RNA-binding protein that promotes renal cell survival upon ischemia [10]. Furthermore, these methods were applied to monitor the impact of ischemia on kidney cell morphology and the distribution of aquaporin-1 and HuR.

_Animal model_

All animal experiments were in accordance with the Animal Care Committee. An established model of murine renal IRI was used [11]. In brief, CD-1 mice were anesthetized (60mg/kg sodium pentobarbital, ip). Kidneys and renal pedicles were exposed by bilateral flank incisions, and renal pedicles were dissected. A non-traumatic vascular clamp was applied across the left pedicle for 30min. The right kidney provided a sham control.

_Tissue samples_

Paraformaldehyde, 16% (Thermo Scientific #28908)

Published methods were applied to examine kidneys from CD-1 mice [11]. In brief, specimens were fixed for 30min in 4% paraformaldehyde, washed three times in PBS and paraffin-embedded, following standard procedures. Specimens were sectioned (4µm sections), placed on gelatin-coated slides and processed.

_Standard equipment for immunohistochemistry_

Glass slide jars (microwave-compatible)

Microwave, sample delimiting pen (DakoCytomation, #S2002) to draw a hydrophobic barrier

Glass coverslips

Mounting medium

_Solutions for deparaffinization and hydration_

Xylene

100% ethanol

95% ethanol in phosphate buffered saline (PBS)

70% ethanol in PBS

50% ethanol in PBS

30% ethanol in PBS

_Antigen retrieval solution_

10mM Trisodium citrate pH 6.0 in water, supplemented with 0.5% (v/v) Tween-20

_Blocking solution_

PBS containing 5% fetal bovine serum (FBS), 0.05% Tween-20 (v/v), 1mM NaN₃

_Permeabilization solution_

PBS containing 0.1% Triton X-100 (v/v), 2mg/ml bovine serum albumin, 1mM NaN₃

_Antibodies_

Mouse anti-HuR (Santa Cruz; #sc-5261)

Rabbit anti-aquaporin-1 (Alpha Diognostics)

Cy3-conjugated anti-mouse IgG Jackson ImmunoResearch (715-165-150)

AlexaFluor®488-conjugated anti-rabbit IgG, Jackson ImmunoResearch (711-545-152)

Secondary antibodies were further purified by pre-adsorption to immobilized proteins prepared from mammalian culture cells.

_Nuclear stain_

4’,6-diamidino-2-phenylindole (DAPI; Sigma)

Automated embedder

Leica EG1150 Modular Tissue Embedder

_Confocal microscope_

Zeiss LSM510 or any other appropriate microscope

Day 1

Deparaffinize tissue sections by immersing slides in a Xylene solution (twice; 10min each).
Hydrate tissue sections by immersion in descending alcohol concentrations. 100% ethanol (10min)

100% ethanol (5min)

95% ethanol in PBS (5min)

70% ethanol in PBS (3min)

50% ethanol in PBS (3min)

30% ethanol in PBS (3min)

Wash slides in PBS (twice; 5min each).
In glass jar, immerse slides in 10mM sodium citrate pH6.0/Tween-20 to retrieve antigens. Place glass jar in center of the microwave (high power setting).

Microwave twice, 3min each for each step.

Make sure tissue sections remain submerged in the solution throughout the procedure.

Allow solution to cool down to room temperature (30-45min).
Remove slide from jar. Draw hydrophobic borders with a Dako-pen around each tissue section.
Add 0.1% Triton-X100 solution to permeabilize cell membranes (5min; room temperature).
Block unspecific binding sites with 5% FBS/Tween-20 solution (1h; room temperature).
Dilute antibodies against HuR (1:250) and aquaporin-1 (1:125) in blocking solution.
Transfer slides to a humidified chamber and add 50-100µl of primary antibodies diluted in blocking solution (overnight; room temperature).

Day 2

Wash with blocking solution (three times; 5min each).
Dilute secondary antibodies in blocking solution (1:500 for Cy3- conjugated anti-mouse IgG; 1:250 for AlexaFluor®488-conjugated anti-rabbit IgG).
Incubate slide with diluted secondary antibodies in a humid chamber (2h; room temperature). Protect from light.
Wash with blocking solution (three times; 5min each).
Stain DNA with 1µg/ml in blocking solution (2min).
Add mounting medium, glass coverslip and seal with nail polish.

Negative controls

Incubate one section on each slide without primary antibodies; add secondary antibodies as described above.
To assess autofluorescence, process control samples without addition of primary or secondary antibodies.

The specific incubation time for each step is included in the procedure section. The protocol is completed on two consecutive days. The time required is _{2h 15min on day 1 and}2h 30min on day 2.

1. Low immunofluorescence signals.

(a) Optimize antigen retrieval; requirements may vary for different antibodies. Consider longer microwave heating, different pH for antigen retrieval, or alternative antigen retrieval buffer.

(b) After step 11, protect slides from light to minimize bleaching of the fluorophore.

2. Uneven staining.

(a) Never let tissue sections dry out; make sure that specimens are always well-covered with the appropriate solution.

(b) For step 4, place a second jar with antigen retrieval buffer in microwave. Use this solution to replenish evaporated retrieval buffer in jar that contains the slides.

3. High background or unspecific staining.

(a) Reduce nonspecific binding of primary and secondary antibodies. Affinity-purify primary and/or secondary antibodies.

(b) Autofluorescence may result particularly in the emission of green light. Switch to other secondary antibodies that emit light in the far red. Alternatively, reduce autofluorescence with published methods [12] or commercially available kits.

4. Rupture of tissue during processing.

In step 5, provide sufficient time for slides to cool down to room temperature. Rapid temperature changes can destroy the tissue.

Application 1. Effect of ischemia on the distribution of aquaporin-1. Aquaporins control water transport across the plasma membrane in the kidney and other organs (reviewed in [13]). Different aquaporin genes are expressed in the kidney in a cell-type specific fashion [14]. The aquaporin-1 protein is highly abundant in epithelial cells of the proximal tubule. In the proximal tubule, aquaporin-1 concentrates in brush border membranes of the apical domain, which is facing the lumen of the tubule [14]. In Fig. 1, we applied our protocol to locate aquaporin-1 in mouse paraffin-embedded kidney sections of controls (sham) and ischemic kidneys (30min ischemia). Following ischemia, the morphology of tubules was altered, material was found in the lumen of tubules, and aquaporin-1 abundance was similar in the apical and basolateral domains of the cells. These observations are consistent with an ischemia-induced impairment of cell polarization and alteration or loss of the brush border.

Our protocols conserve the tissue organization under normal and disease-relevant conditions. Thus, they are appropriate to track physiological and pathological changes, as caused by ischemia, while preserving the ability to distinguish specific tubule segments.

Application 2. Fig. 2 depicts the immunodetection of HuR, a protein located in the nucleus and cytoplasm, and aquaporin-1 in the glomerular and tubular cells. HuR shuttles between the nucleus and the cytoplasm; this nucleocytoplasmic distribution depends on the physiological state of kidney cells [10, 15]. Accordingly, the staining protocol needs to detect both the cytoplasmic and nuclear pools of the protein. Our protocol accomplishes this task (Fig. 2A, 2B, top row), as HuR was detected in different subcellular compartments and in distinct cell types. The presence of HuR in the nucleus is indicated by the overlap with DAPI staining (Fig. 2B, bottom row). However, HuR was also located in the cytoplasm, especially for cells outside of the glomerulus.

Application 3. Detection of nonspecific fluorescence (Fig. 3). Nonspecific binding of antibodies and autofluorescence of the specimens can contribute to high background signals. With affinity-purified secondary antibodies these background signals were low for our protocol.

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We thank Dr. Tomoko Takano (McGill University) for advice with the fixation and embedding procedure and Cindy Baldwin (McGill University) for performing the IRI experiments and harvesting the organs.

The authors declare no competing interests.

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Immunofluorescence staining of kidney sections to monitor ischemia-induced damage

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Immunofluorescence staining of kidney sections to monitor ischemia-induced damage

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Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

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