agarose embedding of lung tissue for multiplex fluorescence imaging

تصوير الرئة المبتكر باستخدام أقسام اهتزازية مدمجة في الأغاروز

Ex vivo lung tissue slices are extensively used in the study of lung disease1. Current gold standards, particularly in clinical and translational large animal studies, employ hematoxylin and eosin staining coupled with brightfield microscopy and observer-based scoring to assess and grade lung dysfunction2,3. While still a valuable technique, it exhibits limitations in terms of spatial resolution and the number of markers that can be imaged simultaneously. Moreover, lung tissue is required to undergo extensive solvent-based washes, which results in dehydration, shrinkage, and rehydration of the tissue before its final imaging4. This process is not only time-consuming but is also environmentally unfriendly, masks protein epitopes, and may invoke structural tissue changes5,6,7,8. However, for lung tissue, embedding in paraffin prior to sectioning has been a necessity owing to the structural fragility of the lung. In contrast, solid organs such as the brain can be cut using a vibrating microtome (vibratome), both in fixed and fresh tissue9,10,11,12.
To enable vibratome sectioning in lung tissue, a method termed precision-cut lung slices&#160;(PCLS) was established where low melting point agarose is injected into fresh lung tissue via the airways or blood vessels and left to solidify13,14. The agarose in the airways provides sufficient structural support to then allow vibratome sectioning9,14. In rodents, this is simple to achieve as agarose can be injected via the trachea; however, in pig and human tissue, it can be challenging to locate a suitable airway/vessel within a given biopsy15,16. Moreover, even if such an airway/vessel is located, substantial force is usually required to inject the agarose, which may influence subsequent lung morphology17.
To overcome the challenges experienced in paraffin embedding/sectioning/staining and the PCLS workflow, we have established a new processing pipeline for fixed lung tissue. This workflow allows the use of a vibratome for sectioning, can produce thinner slices than in PCLS, and yields slices that do not require antigen retrieval for multiplex fluorescence imaging. This method offers a means of &#34;least processing&#34; to generate lung sections that can be used in advanced light microscopy. Moreover, imaging readouts can be used to demonstrate the spatial relationships of cells and anatomical structures within the lung tissue by capturing the volumetric architecture of the tissue18,19,20,21. This paper describes the protocol to generate these lung sections and demonstrates multiplex stains applied to each of these tissues and how these advanced imaging data can be used for quantification.

تسليط الضوء على المؤلف: دراسة التعبير المكاني للبروتين باستخدام أقسام أنسجة الرئة المضمنة في الأغاروز 

التقسيم الاهتزازي الآلي لأنسجة الرئة المضمنة في الأغاروز للتصوير الفلوري المتعدد

This protocol is particularly useful in large animal models of lung transplantation and regeneration, such as pigs, where we can sample small pieces over time. This technique can help us better understand the spatial biology at play in these models. This technique produces minimally-processed lung slices suitable for immunofluorescence imaging without the need for antigen retrieval, and has been effective with all antibodies tested to date.This procedure can be adapted to cut slightly thicker sections, say around one millimeter, which can be then used in light sheet fluorescence microscopy to study the spatial protein expression in a more three-dimensional volume. Our research is investigating factors influencing lung transplant rejection, and developing therapies to reduce rejection using our pig model. This technique helps understand spatial protein changes affecting transplant outcomes post-surgery.We're currently using this technique to explore the spatial protein expression landscape in our pig lung transplant model. We hope that it will help us to better understand the mechanisms behind our experimental therapies. The key to succeeding here lies in vibratome sightings, high blade amplitudes, and lower force speeds are key things to look at if the sectioning goes badly.

Watch this Scientific Journal Video about Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging at JoVE.com

Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging  

تضمين الأغاروز لأنسجة الرئة للتصوير الفلوري المتعدد

للبدء ، قم بإصلاح أنسجة الرئة الخنازير في 4٪ بارافورمالدهيد في درجة حرارة الغرفة لمدة 48 ساعة. ثم انقل أنسجة الرئة إلى أنبوب مخروطي سعة 50 ملليلتر يحتوي على 30 ملليلتر من PBS مع 0.01٪ أزيد الصوديوم. احتفظ بكوب بلاستيكي ناعم ومقص معقم جاهزا.سخني الأغاروز في الميكروويف حتى يصل إلى نقطة الغليان. ثم قم بتبريده إلى 42 درجة مئوية في حمام مائي. قم بتخزينه في حمام مائي حتى يصبح جاهزا للاستخدام.قم بإزالة خزعة الرئة من محلول PBS Azide . استخدم ملقط ناعم ومقص معقم لتشريح فصوص الرئة بعناية إلى كتل أصغر. بعد ذلك ، قم بقطع منتصف كوب بلاستيكي.صب كمية صغيرة من الأغاروز في قاع الكوب. ضع غطاء أنبوب مخروطي سعة 50 ملليلتر مع إزالة حافته على سطح أغارو. قم بتخزين هذا الإعداد عند أربع درجات مئوية لمدة خمس دقائق للسماح للأغاروز بالتصلب.بمجرد أن يصلب الأغاروز ، أخرج الكوب البلاستيكي من الثلاجة. الآن صب حوالي ملليلتر واحد من الأغاروز السائل على الغطاء. ثم ضع كتلة أنسجة الرئة برفق على الغطاء.بمجرد أن يصلب الأغاروز ، يغمر أنسجة الرئة ببطء في الأغاروز السائل. قم بتبريد الإعداد على حرارة أربع درجات مئوية لمدة 15 دقيقة تقريبا حتى يصبح الأغاروز صلبا. استخدم شفرة حادة لإزالة أي أغاروز زائد يحيط بأنسجة الرئة بعناية.

agarose-embedding-of-lung-tissue-for-multiplex-fluorescence-imaging

Research

JoVE Journal

Medicine

Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging

500 Views.  Lund University. للبدء ، قم بإصلاح أنسجة الرئة الخنازير في 4٪ بارافورمالدهيد في درجة حرارة الغرفة لمدة 48 ساعة. ثم انقل أنسجة الرئة إلى أنبوب مخروطي سعة 50 ملليلتر يحتوي على 30 ملليلتر من PBS مع 0.01٪ أزيد الصوديوم. احتفظ بكوب بلاستيكي ناعم ومقص معقم جاهزا.سخني الأغاروز في الميكروويف حتى يصل إل...

Video: Agarose Embedding of Lung Tissue for Multiplex Fluorescence Imaging  

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

To begin, fix the porcine lung tissue in 4%paraformaldehyde at room temperature for 48 hours. Then transfer the lung tissue to a 50 milliliter conical tube containing 30 milliliters of PBS with 0.01%Sodium Azide. Keep a plastic cup fine forceps and sterile scissors ready.Heat the agarose in a microwave until it reaches boiling point. Then cool it down to 42 degrees Celsius in a water bath. Store it in the water bath until ready for use.Remove the lung biopsy from the PBS Azide solution. Use fine forceps and sterile scissors to carefully dissect the lung lobes into smaller blocks. Next, cut down the middle of a plastic cup.Pour a small amount of agarose at the bottom of the cup. Place the lid of a 50 milliliter conical tube with its rim removed on the agaro surface. Store this set up at four degrees Celsius for five minutes to allow the agarose to solidify.Once the agarose has solidified, remove the plastic cup from the refrigerator. Now pour about one milliliter of liquid agarose onto the lid. Then gently place the lung tissue block on the lid.Once the agarose has semis solidified, slowly submerged the lung tissue in liquid agarose. Refrigerate the setup at four degrees Celsius for about 15 minutes until the agarose becomes solid. Use a sharp blade to carefully remove any excess agarose surrounding the lung tissue.

Vibratome Sectioning of Agarose-Embedded Lung Tissue

To begin, embed the lung tissue in agarose. To prepare the lung tissue for sectioning, first, attach each tissue block to the specimen disc of the vibratome with super glue and add ice to the surrounding bath. Then carefully install the specimen disc into the buffer tray and fill the tray with PBS.Next, set the vibratome parameters, keeping the thickness at 200 micrometers, the frequency at 100 hertz, the amplitude of the blade at 1.3 millimeters, and the forward speed of the blade between 0.02 and 0.03 millimeters per second. Commence the sectioning of the lung tissue. Once enough slices have been created, use a brush to gently lift the tissue slice from the vibratome tray.Transfer the slices to a 24 well plate with PBS azide solution. Preserve the lung slices at four degrees Celsius.

التقسيم الاهتزازي لأنسجة الرئة المضمنة في الأغاروز

Immunostaining of Agarose-Embedded Lung Tissue Sections

Begin by embedding lung tissue and agarose and sectioning it using a vibratome. To the sliced tissues, add 300 microliters of the blocking agent. Incubate the plate at four degrees Celsius on a shaker with gentle agitation for 45 minutes.Next, pipette 300 microliters of the PBS diluted primary antibody into each well. Incubate the plate overnight at four degrees Celsius with gentle shaking. Now gently pipette out the primary antibodies without touching the slice.Wash the slices three times with 400 microliters of PBS. Next, pipette 300 microliters of the dilute secondary antibodies into each well. Remove the antibody solution after cold incubation for 90 minutes.Then add the DAPI and tomato lectin 488 to the wells. Incubate the mixture for 30 minutes. After removing DAPI and tomato lectin, wash the slices in PBS for three cycles of 10 minutes.With a brush, transfer the slices to a slide. Place three drops of mounting medium on the slide and mount a cover slip over it before imaging. The lung sections of mouse, pig, and human samples were immunofluorescently stained for SMA, elastin, and LEA.The SMA and elastin were observed to be distributed across structures such as alveolar ducts, blood vessels, bronchioles, and alveoli. Comparison of type II pneumocytes distribution in adult and infant humans was performed. The type II alveolar cell antibody HT2-280 had a strong affinity to the pneumocytes cell surface.The infant sample yielded a significantly higher type II pneumocytes count. However, the infant sample also exhibited a significantly higher scaffold coverage than the adult. The number of type II pneumocytes based on scaffold coverage was also significantly higher in the infants.