TITLE: Tertiary Lymphoid Structures in Mouse Models of Lung Cancer: From Induction to Imaging
AUTHORS: Wendy Memishian, Teresa McGee, Julie Wells, Carol Bult

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Tertiary lymphoid structures (TLSs) are a recently-described immune response that occurs when chronic inflammation leads to an organ-like organization of immune cells and specialized blood vessels. In humans, they have been shown to develop in and around many different types of tumors, and often lead to a more promising prognosis when found. In non-small cell lung cancer (NSCLC), they have a highly beneficial effect on patients' survival. What remains to be studied is the heterogeneity of the TLSs and the mechanisms of their development. In order to study these important aspects of the immune response to NSCLC, we developed a protocol to image a time course of TLS development in murine bronchiolo-alveolar adenomas and adenocarcinomas, and further expanded on how to quantify, and therefore characterize, these structures.</div></div>

[STEPS]
?. [Perfusion and Harvest of Lungs]
Euthanize mouse using an IACUC-approved method, such as carbon dioxide, until it no longer responds to toe pinches.
?. [Perfusion and Harvest of Lungs]
Weigh mouse before dunking completely in soapy water.
?. [Perfusion and Harvest of Lungs]
Pin down limbs and cut open abdomen from the intestines to the rib cage using surgical scissors.
?. [Perfusion and Harvest of Lungs]
Cut through the diaphragm as a secondary form of euthanasia, being careful not to cut into the liver, lungs, or heart. Continue snipping it all the way across the bottom of the rib cage. Cut completely through the sternum and the rib cage in order to allow complete inflation of the lungs.
?. [Perfusion and Harvest of Lungs]
Find the inferior vena cava and snip it in order to begin perfusion of the lungs .
?. [Perfusion and Harvest of Lungs]
Using a 5 mL syringe with a 23g needle, find the right ventricle of the heart and slowly inject 1XPBS until lungs inflate completely.
[1XPBS]
?. [Perfusion and Harvest of Lungs]
To fix lungs, prepare a new 5 mL syringe filled with 10% neutral buffered formalin and a new 23g needle. Using the surgical scissors, snip soft tissue away from the throat area until the trachea is in view. Holding the needle parallel to the trachea, insert the needle with the bevel up, careful to enter the trachea and not go completely through it. Slowly inject all of the fixative and hold the needle in the trachea for at least 1 minute afterwards to ensure all of the lung gets contact with the 10% NBF.
[10% NBF]
?. [Perfusion and Harvest of Lungs]
After removing the needle, grab the trachea gently with forceps and cut above where it is held. Lift the trachea and, holding the scissors parallel to the body, snip away tissue attaching the trachea, thymus, heart, and lungs to the body. Immediately place the organs in a 50 mL conical tube filled with 10% NBF.
[10% NBF]
?. [Perfusion and Harvest of Lungs]
Leave the organs in 10% NBF overnight. Move to 1XPBS the following day.
[1XPBS]
?. [Sectioning and Staining of Lungs]
Put organs into a dish with some of the 1XPBS and, using a dissecting scope, cut or pull the trachea, thymus, and heart from the lungs. Then separate the 5 lobes of the lungs, removing as much connective tissue as possible without damaging them.
?. [Sectioning and Staining of Lungs]
Move the lung lobes from the dish into a slotted tissue cassette, making sure to spread the lobes out as much as possible to ensure good staining and imagine. Put the cassettes into 70% ethanol.
[70% EtOH]
?. [Sectioning and Staining of Lungs]
Paraffin embed the tissues. Wait to begin sectioning until all future materials are prepared, as antigens degrade once sectioning begins.
?. [Sectioning and Staining of Lungs]
Serial section the paraffin block into 5um sections, preferably with one section per slide, but up to three per slide.
?. [Sectioning and Staining of Lungs]
Stain every fourth slide with hematoxylin and eosin (H&E) in order to find "levels" of interest in the block (ex. stain the first slide with H&E, if it appears interesting, then the first level, consisting of slides 2, 3, and 4, will proceed to the rest of the stains).
?. [Adenovirus Infection]
At 6-7 weeks of age, mice are separated by sex and treatment group (i.e. female control, female experimental, etc.).
?. [Sectioning and Staining of Lungs]
For each level of interest, perform IHC on the second slide with anti-CD3/CD20 dual staining, on the third slide with anti-PNAd, and on the fourth slide with anti-CD11c.
?. [Sectioning and Staining of Lungs]
Look for hyperplasia, adenomas, and adenocarcinomas.
?. [Sectioning and Staining of Lungs]
Around the abnormal cells, look for areas of lymphoid aggregates that are peritumoral or intratumoral (lymphoid aggregates near vessels in the lungs are common with aging mice, and are not a result of the tumor).
?. [Adenovirus Infection]
Mice are given an intraperitoneal injection of tribromo-ethanol (Avertin) at a dosage of 0.02 mL/g of body weight, which should render the mouse unable to respond to a toe pinch.
?. [Adenovirus Infection]
Combine MEM, CaCl2, and  Ad5mSPC-Cre (of a known titer) to get 2.5 EE8 PFU. Let the combination sit for 20 minutes.
?. [Adenovirus Infection]
Using a pipet, have the mouse breathe in 25 uL of the virus by holding droplets against one nostril slowly. After ~5 min, have the mouse breathe in another 25 uL through the other nostril in droplets.
?. [Adenovirus Infection]
Lay the mouse on its side. Put the pen over a heating pad or under a heat lamp in order to keep the mouse's temperature up while it's anesthetized. Observe to make sure all mice wake up and are able to easily get food and water before putting back on the shelf.
?. [Sectioning and Staining of Lungs]
Scan the slides and use software (ImageJ, CellProfiler, etc.) to overlay the images of the different parts of the TLS, showing a bona fide TLS.