Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Podsumowanie

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Streszczenie

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

Wprowadzenie

Adipose tissue has key metabolic and endocrine functions¹. It dynamically expands or shrinks in response to different nutrient stresses². The active tissue remodeling process consists of multiple physiological paths/steps including angiogenesis, fibrosis, and shaping of local inflammatory microenvironments^2,3,4. Some physical stimuli, such as cold exposure and exercise, may trigger sympathetic activation, which ultimately leads to new blood vessel formation and sympathetic innervation in adipose tissues^5,6. These remodeling processes are linked tightly to systemic metabolic outcomes including insulin sensitivity, the hallmark of type 2 diabetes². Thus, visualization of these pathological changes is very important to understanding the healthy status of whole adipose tissues.

Angiogenesis is the process of new blood vessel formation. Since blood vessels provide oxygen, nutrients, hormones, and growth factors to tissue, angiogenesis has been considered a key step in adipose tissue remodeling, which has been documented with different techniques^{6,7,8,9,10,11,12,13}. However, there remain questions about the resolution of the images, efficiency of immunostaining, and methods for quantification of vessel density. Compared to new blood vessel formation, innervation in adipose tissue has been underestimated for a long time. Recently, Zeng et al.¹⁴ used advanced intravital two-photon microscopy and demonstrated that adipocytes are surrounded by layers of nerve fibers¹⁴. Since then, researchers have started to appreciate the pivotal role of sympathetic innervation in regulation of adipose tissue physiology. Thus, developing an easy and practical approach to document adipose nerve innervation is important.

Here, we report an optimized method for the co-staining of blood vessels and nerve fibers based on our previous protocols. With this method, we can achieve clear images of blood vessels and nerve fibers without noisy background. Moreover, we obtain a resolution that is high enough for performing quantitative measurement of densities with open source software. By using this new approach, we can successfully compare the structures and densities of blood vessels and nerve fibers in different adipose depots.

Protokół

All procedures containing animal subjects have been approved by the Animal Welfare Committee of University of Texas Health Science Center at Houston (animal protocol number: AWC-18-0057).

1. Reagent Preparation

1. 1x phosphate-buffered saline (PBS, pH 7.4): to make 1 L of 1x PBS, dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g of KH₂PO₄ in 800 mL of distilled water. Adjust the pH to 7.4 and fill with distilled water to achieve a final volume of 1 L.
2. 1% paraformaldehyde in 1x PBS (1% PFA, wt/vol): to achieve a final volume of 50 mL, add 3.125 mL of 16% PFA (see Table of Materials) to 46.875 mL of 1x PBS. Mix well and store at 4 °C for later use.
   CAUTION: Paraformaldehyde is toxic. All procedures should be carried out under a fume hood to avoid inhalation and skin contact.
3. 0.1% polysorbate 20 (see Table of Materials) in 1x PBS (1x PBST, pH 7.4, vol/vol): to achieve a final volume of 50 mL, add 0.05 mL of polysorbate 20 to 49.95 mL of 1x PBS. Vortex and store at 4 °C for up to 1 month.
4. 1% octoxynol-9 (see Table of Materials) in 1x PBS (1x PBS-TX, pH 7.4, vol/vol): to achieve a final volume of 10 mL, add 0.1 mL of octoxynol-9 to 9.9 mL of 1x PBS. Vortex and store at 4 °C for up to 1 month.
5. 2% sodium azide (wt/vol): to produce 10 mL of 2% sodium azide, add 0.2 g of sodium azide to 10 mL of 1x PBS. Mix well and store at 4 °C for later use.
6. 90% glycerol in 1x PBS (vol/vol): to achieve a final volume of 10 mL, add 1 mL of 1x PBS to 9 mL of glycerol. Vortex and store at room temperature (RT) for later use.

2. Animal Ddissection and Adipose Tissue Collection

1. Use 6-week-old C57BL/6J male mice. Anesthetize the mice with isoflurane (4%–5% for induction then 1%–2% for maintenance). Once anesthetized, perform a toe-pinch reflex to determine the anesthetic depth, judging by the lack of pedal reflexes. Once the mice are deeply anesthetized, dissect the mice following the protocol as previously published¹⁵.
2. Sterilize the blunt scissors and surgical area of the chest (no need to shave the hair). Open the chest by cutting the diaphragm and ribs along the lateral surface with a size of ~2 cm using blunt scissors. Clamp the chest flag with hemostat and reflect it by putting the hemostat over the head.
3. Make a small incision into the left ventricle (~0.5 cm) with iris scissors. Insert an olive-tipped perfusion needle through the incision site and clamp the needle tip in place with the hemostat. Attach the needle to a 50 mL syringe containing paraformaldehyde fixation solution (1% PFA, pH 7.4).
4. Open the right atrium by section cutting with iris scissors as the perfusion outlet. Start the perfusion by gently and continuously pushing the syringe at a perfusion rate close to 10 mL/min. Stop pushing when the fluid exiting the outlet is clear of any blood. The whole process should require ~5 min.
   CAUTION: Perform these steps under a fume hood to prevent toxicity from PFA.
5. Dissect white and brown adipose tissues from the mice. Use scissors to break the tissue samples into small pieces of chunks approximately 5 x 5 x 3 mm³. Transfer the small chunks with the sterilized tweezers into the tissue embedding cassettes with proper labeling of the tissue samples on the cassettes.
6. Immerse the embedding cassettes containing tissue samples into the fixation solution (1% PFA in 1x PBS, pH 7.4) at 4 °C for 24–48 h.
   NOTE: The fixation solution and fixation time vary based on sizes of the tissues^16,17,18.
7. Wash the samples with fresh 1x PBS buffer three times (0.5 mL/wash).
   CAUTION: Perform this step under a fume hood. The paraformaldehyde waste should be properly handled according to hazardous waste disposal procedures.
   NOTE: The tissues can be stored in 1x PBS at 4 °C for further processing.

3. Antibody Incubation

1. Cut the fixed tissue samples into approximately 2 mm³ cubes with the sterilized scissors. For permeabilization, transfer the samples into a 1.5 mL tube containing 1 mL of 1x PBS-TX (1% octoxynol-9 in 1x PBS, see Table for Materials, pH 7.4) and gently rotate the tubes at RT for 1 h at 18 rpm.
2. Carefully remove the 1x PBS-TX by aspiration. Wash the samples 3x by adding the 1x PBS directly into the same tubes (no need to change the tubes). During each wash, invert the tubes several times.
3. For blocking, add 0.5 mL of blocking buffer (Table of Materials) to the samples and incubate at RT for 2 h with gentle rotation.
4. To prepare 0.4 mL of primary antibody solution, dilute 2 μL of anti - endomucin (the marker of blood vessels) ⁵ (1:200) and 2 μL of anti — tyrosine hydroxylase (TH, the marker of nerve fibers) ⁵ (1:200, 1 µg/mL) antibodies (Table of Materials for antibody information) into 396 μL of blocking buffer. Vortex and spin down to recover the volume.
5. For the first antibody incubation, carefully remove the blocking buffer from the tissue chunks. Add 100 μL of primary antibody solution prepared in step 3.4 into tubes and incubate at 4 °C overnight.
   NOTE: The antibody dilution and incubation time may vary among different antibodies. It is recommended to add 0.02% sodium azide to the antibody solution to prevent microbial growth. To prepare 0.4 mL of primary antibody solution with sodium azide, add 4 μL of antibodies (1 μL of each) and 4 μL of 2% sodium azide into 392 μL of blocking buffer.
6. Carefully collect the primary antibody solution for reuse if desired. Wash the samples with 1x PBST (pH 7.4, 100 µL/wash) three times (30 min each) with gentle rotation at 18 rpm.
7. For the preparation of secondary antibody solution, dilute 2 μL of fluorophore (wave length 495 nm) conjugated anti-goat IgG (1:200) and 2 μL of fluorophore (wave length 650 nm) conjugated anti-rabbit IgG (1:200) (see Table of Materials) into 396 μL of blocking buffer. Vortex and spin briefly to collect all the liquid.
   NOTE: Protect the samples from light during the following procedures.
8. For the second antibody incubation, remove the last wash buffer from samples. Add 100 μL of secondary antibody solution into tubes and incubate at RT for 2 h with gentle rotation at 18 rpm.
9. Carefully remove the secondary antibody solution. Wash the samples 3x with 1x PBST (pH 7.4, 30 min each) with gentle rotation at 18 rpm.
   NOTE: Do not reuse this secondary antibody solution, as it may be contaminated with the trace amounts of primary antibodies brought by the tissues.
10. For optical clearance, immerse the samples in 1 mL of 90% glycerol and keep the samples at 4 °C in darkness until they become transparent.
    NOTE: The immersion duration depends on the tissue type and size. Typically, a 2 mm³ cube of white adipose tissue needs overnight incubation, while the same size brown adipose tissue sample needs longer.
11. Adhere a silicone isolator to the slide to create a well for volume imaging. Alternatively, attach several layers of transparent tape to the slides and cut a square out of the middle to create a well (Figure 2).
    NOTE: Adjust the well depth to fit the sample size. In this protocol, a 1 mm well depth is used.
12. Carefully transfer the samples into the well and fill it with the mounting medium (see Table of Materials). Lay a cover slip on the surface and seal the corners of the cover slip with high viscosity medium (see Table of Materials). Let the mounting medium cure for 24 h at RT in darkness.
    NOTE: Ensure that no space is left between the sample and cover slip. Avoid bubbles under the cover slip. Avoid placing excessive pressure on the samples.

4. Image Acquisition

1. Acquire Z-stack images (refer to steps 4.2–4.7) with the 20x objective of a confocal microscope/software.
2. Start the system. Click on the <Configuration> tab and activate the argon laser and HeNe 633 laser. Click on the <Acquire> tab. In the <Visible> laser lines panel, move the corresponding intensity sliders up to choose the laser lines of 488 nm and 633 nm. Initially, the intensities can be set to 20%–30%. Select the 488/561/633 triple dichoric mirror. Activate the PMTs by clicking on the <Active> checkboxes, choosing PMT1 for the emission excited by the 488 nm laser and PMT3 for that of the 633 nm laser. Set the wavelength range to 500–550 nm for PMT1 and 650-750nm for PMT3. Select the psudocolor to be used for image display by double clicking the colored rectangle beside each PMT and choosing a color from the pop-up menu.
3. Click on <Live> to check the image of samples. Use the z-Position knob to select a plane of focus within the XY region of interest. Adjust the brightness of the images by tuning the laser intensity, smart gain and offset. For each fluorescence channel, perform this adjustment under the <QLUT> (Quick Look Up Table) display mode. Click on the <QLUT> button to enter the QLUT mode, in which saturated pixels are displayed in blue to aid the setting of appropriate brightness level. Click twice on the <QLUT> button to go back to the pseudocolor display mode.
   NOTE: The numerical values of the setting may vary among each experiment.
4. While scanning, turn the Z-Position knob counterclockwise to move the plan of focus to one end of the volume of interest and then click on the <End> arrowhead to set the scanning end position. Then turn the Z-Position knob clockwise to move the plane of focus through the specimen to the other end of the volume of interest and then click on the <Begin> arrowhead to set the scanning begin position.
   NOTE: For the comparison between different samples, define the same Z-volume for different samples.
5. Adjust the z-step size to 3 µm. Change the image quality by selecting a format of 1024 x 1024, speed of 100 Hz, and line average of 2.
6. Select <Start> to initiate the z-stack image acquisition.
7. For maximum projection of the acquired image stack, click on <Process> tab and then <Tool>, select <3D Projection> and enter <Maximum> in the method list without changes to X, Y and Z. Set <Threshold> to 0. Click on <Apply>. The maximum intensity of the Z-volume will be stacked into a 2D image which will be shown (Figure 4A).

5. Analysis of Blood Vessel and Nerve Fiber Networks

1. Analysis of 2D images via open source software (see Table of Materials)¹⁹
   1. Open the stacked images in the software. Adjust Vessel diameter and intensity until all the vessel structures can be properly selected (Figure 4B).
   2. Select Run analysis and export the data following guidance of the software.
2. Analysis of 3D images via the licensed software (see Table of Materials)
   1. Open the raw data of images with the software. Adjust the threshold and other parameters until the vessel structures in each layer of the z-volume are properly selected (refer to the user guide for details in the software) (Figure 4C).
   2. Analyze the selected segments characters and export the data following the guidance of the software.

Wyniki

The distal region of the epididymal white adipose tissue (eWAT), medial region of the dorsolumbar subcutaneous white adipose tissue (sWAT), and medial region of the interscapular brown adipose tissue (BAT) were collected. The locations for collecting these tissues are indicated in Figure 1.

Figure 1: Ana...

Dyskusje

Adipose tissue remodeling is directly linked to metabolic dysregulation during obesity development^1,2. Angiogenesis and sympathetic innervation are both essential for the dynamic remodeling process^2,12. Therefore, developing an applicable approach to visualize the new blood vessels as well as nerve fibers are of great importance. Previous methods have been reported for documenting angiogenesis in adipose ...

Materiały

Name	Company	Catalog Number	Comments
Alexa Fluor 488 AffiniPure Bovine Anti-Goat IgG (H+L)	Jackson ImmunoResearch	805-545-180	Lot: 116969
Alexa Fluor 647 AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch	711-605-152	Lot: 121944
Amira 6.0	Thermo Fisher Scientific		Licensed software
Angio tool	National Institutes of Health		Open source software https://ccrod.cancer.gov/confluence/display/ROB2/Home
Anti-mouse endomucin antibody	R&D research system	AF4666	Lot: CAAS0115101
Anti-tyrosine hydroxylase antibody	Pel Freez Biologicals	P40101-150	Lot: aj01215y
Cover glasses high performance, D = 0.17 mm	Zeiss	474030-9020-000
Cytoseal 280	Thermo Fisher Scientific	8311-4	High-viscosity medium
Glycerol	Fisher	G33-500
Paraformaldehyde,16%	TED PELLA	170215
Press-to-Seal Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 1.0 mm deep	INVITROGEN	P24744	Silicone isolator
ProLong Diamond Antifade Mountant	Thermo Fisher Scientific	P36965	Mounting medium
SEA BLOCK Blocking Buffer	Thermo Fisher Scientific	37527X3
Sodium azide	Sigma-Aldrich	S2002-100G
Tissue Path IV Tissue Cassettes	Thermo Fisher Scientific	22-272416
Triton Χ-100	Sigma-Aldrich	X100	Generic term: octoxynol-9
Tube rotator and rotisseries	VWR	10136-084
Tween-20	Sigma-Aldrich	P1379	Generic term: Polysorbate 20