Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Information on all material used is given in Table 1. All animal experiments were performed under the authority of a U.K. Home Office project license.

1. CatnB Deletion using the Ah-cre and vil-Cre-ER^T2 Systems

1. Cross the mice strains to generate 10 – 14 week old cohorts of Ah-cre⁺CatnB^+/+, Ah-cre⁺CatnB^flox/flox, vil-Cre-ER^T2CatnB^+/+ and vil-Cre-ER^T2CatnB^flox/flox.  For visual analysis of recombination, via a β-gal­ stain, cohorts should contain the Rosa26R-lacZ  reporter¹⁷.  Cohorts should control for the presence of modified genes and use of induction agents, the size of cohorts required should be estimated using a power analysis.
2. To induce the Ah-cre transgene prepare β-Naphthoflavone (BNF), or for the vil-Cre-ER^T2 transgene tamoxifen (TAM) in corn oil to give a working solution of 10 mg/ml. NOTE: The agents should be weighed out in a fume hood using appropriate personal protection.
3. Heat solutions in an amber bottle (BNF is light sensitive) to either 99.9 °C for BNF or 80 °C for TAM in a water bath.
4. Transfer to a heated stirrer set at 100 °C for BNF, or 80 °C for TAM, and stir for 10 min.
5. For BNF repeat 1.3-1.4 until dissolved (can take >1 hr).
6. Aliquot into small amber bottles (~5 ml) and then freeze at -20 °C.  Bottles should be discarded after 3 freeze/thaw cycles.  Prior to use thaw agents and reheat to appropriate temperature if they have fallen out of solution.  Allow to cool to <37 °C prior to injection.
7. Inject the mice intraperitoneally (I.P.) with a dose of 80 mg/kg, for example a 25 g mouse receives 0.2 ml of the appropriate induction agent. For Ah-cre deliver three injections in 24 hr period, for vil-Cre-ER^T2 give one injection per day for 4 days.

2. Dissection of Intestine for Reporter Visualization and Immunohistochemistry (IHC)

1. Euthanize the mouse using cervical dislocation without prior anesthesia in accordance with ethical approval. Place mouse in a supine position and wet the fur using 70% EtOH, open the intra-peritoneal cavity longitudinally along the midline using a scissors.
2. Secure the stomach with a forceps and sever the connection to the esophagus. Remove the small intestine up to the appendix by gently pulling on the stomach. Remove the large intestine up to the anus by gently pulling on the appendix.
3. Once the intestines have been isolated remove the stomach and appendix. Flush intestines with 1x PBS using a syringe with a blunt ended pipette tip.  NOTE: Each intestine should be immediately processed for one of the downstream applications described below.

3. Formalin Fixation of Intestine

1. Cut the flushed intestine into 3 equal sized sections and label proximal, middle and distal. Cut each section into 1 cm pieces. Take a small strip of surgical tape 2 cm x 2 cm. Place three to five 1 cm pieces on to the middle of the surgical tape in a pyramid formation.
2. Close and seal the tape around the pieces longitudinally, to give a “log pile” effect. Place tissue in a flat bottomed container containing a large excess of neutral buffered formalin fixative, at least 10x the volume of fixative to the volume of tissue.  NOTE: Avoid placing excess amounts of tissue within a tube for fixation, divide it into multiple containers.
3. Place samples at 4 °C for at least 18 – 24 hr prior to embedding and sectioning.  To prevent loss of nuclear β-catenin, do not fix beyond 24 hr. After fixation transfer tissue to a flat bottomed container containing a large excess of 70% EtOH, at least 10x the volume of tissue.

4. Methacarn Fixation of Intestine

1. Prior to dissection prepare methacarn fixative by combining 300 ml MeOH, 150 ml chloroform and 75 ml glacial acetic acid (4:2:1). Cut the flushed intestine into 3 equal sized sections proximal, middle and distal.
2. Place each piece of intestine side by side on a piece of filter paper (15 cm x15 cm) and using a springbow scissors open it up ‘en face’. Place the intestine and filter paper into a glass dish containing methacarn for 3 – 24 hr at RT. After fixation pick up the end of an intestine section using a forceps.
3. Wind the intestine around the forceps to form a “swiss roll” and secure the roll by slightly opening the forceps and putting a 25 G needle through it.   Place tissue in a flat bottomed container containing a large excess of neutral buffered formalin fixative, at least 10x the volume of fixative to the volume of tissue and store for at least 1 hr before proceeding to processing.

5. Whole Mount LacZ Visualization (Modified from El Marjou et al.¹⁸)

1. Prepare wax plates by combining molten ralwax with mineral oil at 10:1. Pour into 15 cm petri dishes and leave to cool. Prepare X-gal fixative as per Table 1 and store on ice.
2. Remove whole intestine and flush through with ice-cold 1x PBS, as per section 2. Fix intestine by flushing with 25 ml ice-cold X-gal fixative.
3. Using a scissor cut the intestine into 3 – 5 equal sections (maximum 5 per plate).  Place each section onto wax plate and pin down each end so the section is slightly stretched with the mesenteric line uppermost; trim any excess mesentery. Using a springbow scissors cut the gut longitudinally and pin out along the way. 
4. Flood the plate with X-gal fixative to cover the sections and leave for at least 1 hr at 4 °C. Remove X-gal fixative using a 25 ml pipette and wash once with 30 ml of 1x PBS. Cover sections with 30 ml of DTT demucifying solution for 30 – 60 min at RT, ideally on a rocking platform.
5. Remove demucifiying solution using a 25 ml pipette and flood the plate with 30 ml of 1x PBS.  Using a pasteur pipette wash the intestine sections with the 1x PBS in the plate to remove mucus.
6. Remove 1x PBS with a 25 ml pipette and flood with 30 ml of X-gal stain. Incubate overnight at RT in the dark with gentle agitation on a rocking platform.
7. Following the overnight incubation check that the sections have developed a blue/green stain, if the background color is still white, fresh staining solution can be added and monitored till staining develops. NOTE: Once sections have stained then no further staining can be attempted.
8. Remove the X-gal stain using a 25 ml pipette and flood plate with 30 ml of 1x PBS and leave for 3 min with gentle agitation.  Remove pins and pick up, with forceps, the end of an intestine section. Wind the intestine around the forceps to form a “Swiss roll”, secure the roll by slightly opening the forceps and putting a 25 G needle through it.
9. Place tissue in a wide mouthed flat bottomed container containing a large excess of neutral buffered formalin fixative, at least 10x the volume of fixative to the volume of tissue.   Place samples at 4 °C for at least 24 hr prior to embedding and sectioning. 

6. Extraction of Crypts from Intestine

1. Isolate the first 20 cm of the small intestine, as per section 2. Place the intestine on a clean dissecting surface and using a forceps and scissors remove any attached fat/mesentery. Using a springbow scissors open the gut longitudinally.
2. Using a microscope cover slide, firmly scrape the gut lumen to remove villi and mucus. Using a scissors cut the intestine into ~5 mm pieces and transfer into a 50 ml tube with 25 ml 1x HBSS supplemented with penicillin (100 U/ml) and streptomycin (100 U/ml). 
3. Incubate for 10 min at RT. Remove the antibiotic containing media by passing the samples through a 70 µm cell strainer.
4. Place intestinal sections into a fresh 50 ml tube containing 10 ml 1x HBSS and replace the cap.
5. Gently invert twice and remove the 1x HBSS by passing through a 70 µm cell strainer. Further wash the intestinal pieces by repeating 6.4 & 6.5  three times and ensure that the final fraction is relatively clear.
6. Transfer tissue to a fresh 50 ml tube containing 10 ml of EDTA (8 mM)/1x HBSS and leave at RT for 5 min. Shake vigorously (20 – 30x) or vortex, pass through a 70 µm cell strainer and transfer tissue pieces to a fresh 50 ml tube containing EDTA (8 mM)/1x HBSS.  NOTE: The flow through can either be discarded or retained if analysis of villi epithelia is required.
7.  Incubate the tissue pieces on ice for 30 min, shake the sample vigorously (20 – 30x) or vortex. Pass the samples through a 70µm cell strainer and retain the flow through as this contains the crypts.
8.  Transfer the tissue pieces to a fresh 50 ml tube containing 10 ml of 1x HBSS.  Shake vigorously (20 – 30x) or vortex, pass through a 70 µm cell strainer and retain the flow through. Repeat one more time to ensure maximum recovery of crypts from intestinal pieces. Combine the flow through fractions and centrifuge at 300 x g for 5 min. Pour off the supernatant and retain the crypt pellet.  NOTE: The pellets can be used immediately for culturing (if applicable) or stored at -80 °C prior to standard DNA/ RNA/protein extraction procedures.

7. Standard Immunohistochemical Visualization

1. Cut 5 µm sections of paraffin embedded tissue onto poly-L-lysine (PLL) slides. Note: A standard protocol for staining with a B-catenin antibody is given below, parameters for other antibodies are given in Table 2.
2. De-wax with 2x 3 min washes in slide baths containing fresh xylene. Rehydrate by passing slides for 3 min through slide baths containing fresh: 100% EtOH (2x), 95% EtOH and 70% EtOH and finally into 1x PBS.
3. Place slides into a slide bath containing citrate buffer (pH 6) and heat 99.9°C for 20 min to retrieve antigens. Allow slides to cool and then wash 3x 5 min in a slide bath containing 1xTBS/T for 5 min. Remove slides from last wash, immediately draw around tissue with a PAP pen, and cover section with a commercial peroxidase block or 1.5% H₂O₂ (in distilled H₂O).
4. Incubate for 20 min at room temperature (RT) then wash 3x in slide baths containing fresh 1x TBS/T for 5 min. After washing cover sections, using a pipette, in 5% Normal Rabbit Serum (NRS)/1xTBS/T for 30 min at RT to block non-specific hydrophobic binding of your primary antibody.
   1. Remove NRS block with a Pasteur pipette and cover section in B-catenin primary antibody diluted 1:200 with 5% NRS. Wash slides for 3x 5 min in slide baths containing fresh 1xTBS/T. Note – other antibodies will require optimization for dilution and specificity.  To ensure specificity of an antibody, appropriate no antibody and isotype control stains should be performed.  An isotype control is matched to the host species and isotype of your primary antibody.
5. Visualize with either a commercial HRP detection kit or with an appropriate fluorescently labelled secondary antibody (Table 2).  NOTE: Length of development differs for each antibody, for β-catenin 10 – 15 sec is usually sufficient.  To optimize development for an antibody first establish the length of time required to visualize positive cells using a positive control slide and then apply this to all subsequent slides.
6. Wash slides for 3x 5 min in slide baths containing fresh TBS/T. Counterstain slides by immersing in a slide bath containing haematoxylin for ~45 sec (not required if using fluorescently labelled secondary antibodies).  Put slides into a clean slide bath and rinse with running tap water for ~1 min, ensuring the haematoxylin is not completely washed out.
7. Dehydrate slides by passing through slide baths containing increasing concentrations of alcohols; 1x 30 sec  in 70% EtOh, 1x 30 sec in 95% EtOH, 2x 30 sec washes in 100% EtOH, 2x 2 min in xylene.
8. Mount slides under a coverslip using a commercial mounting media. NOTE: If using fluorescently labeled antibody mount with a media containing DAPI to label the nucleus.

8. Histological Identification of Specific Intestinal Epithelial Cells

1. Enterocytes
   1. Prepare sections using section 7 with the villin antibody and conditions described in Table 2.
2. Entero-endocrine cells (Grimelius stain ^18,19).
   1. Prepare sections by following steps 7.1-7.2. Wash slides in a slide bath containing ultrapure water for 3 min. Transfer the slides to a slide bath containing preheated silver solution (Table 1) and incubate at 60 °C for 3 hr.
   2. Remove the slides from the silver solution and place in a slide bath containing freshly prepared preheated reducer solution (Table 2) at 45 °C for 3 min.  Remove the slides and place into a slide bath containing fresh ultrapure water for 3 min. Follow steps 7.6-7.8 from section 7.
3. Goblet cells (Alcian Blue stain).
   1. Prepare sections by following steps 7.1-7.2.. Transfer slides to a slide bath containing Alcian Blue pH 2.5 for 5 min at RT.  Remove Alcian blue stain with a pipette and place slide bath under a running tap for 3 – 5 min. Follow steps 7.6-7.8.  NOTE: Alcian blue solution can be retained for further use.
4. ISCs.
   1. To identify the ISCs follow section 9.
5. Paneth cells.
   1. Prepare sections following section 7 using the lysozyme antibody and conditions described in Table 2.

9. In Situ RNA Detection with the Murine Intestine^19-21

1. Place 5 µm sections from formalin fixed intestine (section 3) onto PLL slides.   Prepare a linearized digoxigenin labelled RNA probe for detecting Olfm4 expression²¹. Dewax and rehydrate sections as per section 7. NOTE: This protocol should be performed in an RNAse free environment to prevent degradation of the RNA probe.
2. Draw around tissue with a PAP pen to minimize reagents. Incubate sections in a slide bath with 6% H₂O­₂ (in distilled H₂O) for 30 min.  Wash twice in a slide bath with fresh 1x PBS for 3 min. Discard 1x PBS and, using a pipette, cover section with 4% paraformaldehyde for 20 min on ice.
3. Wash twice in a slide bath with fresh 1x PBS for 3 min. Using a pipette cover sections with Proteinase K solution for 5 min  Wash in a slide bath with fresh 1x PBS for 3 min. Using a pipette post-fix sections by covering in 4% paraformaldehyde for 5 min at RT.
4. Wash in a slide bath with DEPC treated H₂0 for 2 min. Using a pipette cover sections in acetic anhydride solution for 10 min with agitation. Wash in a slide bath with fresh 1x PBS/3 min, followed by 1x saline/3 min. Pass slides through baths containing increasing concentrations of alcohols; 1x 30 sec  in 70% EtOh, 1x 30 sec in 95% EtOH, 2x 30 sec washes in fresh 100% EtOH, 2x 2 min in fresh xylene and allow to air dry.
5. Dilute Olfm4 probe 1:100 in hybridization buffer and denature probe by heating to 80˚C for 3 min. Apply 100 μl of probe to each section and cover with parafilm to prevent dehydration of the slide.  Incubate overnight in a dark, moist chamber at 65 ˚C.
6. Wash in a slide bath with 5× SSC at 65 ˚C for 15 min. Wash sections in a slide bath twice with fresh 50% formamide/5× SSC/1% SDS for 30 min at 65 ˚C. Wash sections twice in a slide bath in fresh PBT for 10 min, the first at 65 ˚C and the second at RT. Using a pipette cover sections with PBT containing 25 µg RNAse for 45 min at 37 ˚C. Wash sections in a slide bath in PBT for 5 min at RT.
7. Wash sections in a slide bath twice with fresh 50% formamide/5× SSC for 30 min at 65 ˚C. To block, cover sections using a pipette with 10% sheep serum in PBT and store in a dark, moist chamber at RT for 2 – 3 hr.
8. Prepare antibody by diluting anti-digoxigenin alkaline phosphatase conjugated antibody at 1:500 with 10% sheep serum in PBT containing 5 mg/ml mouse intestinal powder. Incubate for 3 hr at 4 °C in dark on a rocking platform. Spin down to remove excess intestinal powder and add 3x volumes of 1% sheep serum in PBT to the supernatant.
9. Remove block from slides with a pipette and add 100 μl of the antibody solution to each section, cover with parafilm and incubate in a dark, moist chamber at 4°C overnight. Wash sections in a slide bath 3× with fresh PBT for 5 min. To block sections wash in a slide bath 3× with fresh NTMT buffer for 5 min.
10. To visualize, using a pipette cover each section with BM purple and incubate in the dark at RT for 24 – 72 hr until a sufficiently strong colour develops. Wash sections in a slide bath once in PBT and counterstain by immersing in eosin for 1 min.  Remove excess eosin by washing sections in a slide bath under a running water for 3 – 5 min. Immerse slides in xylene and allow to air dry. Mount under a coverslip using commercial media.

10. Histological Characterization of Intestinal Epithelia

1. Place 5 µm sections from the fixed tissue (section 3 & 4) onto PLL slides.  Analyze ≥25 random whole (or ≥50 half) crypts from ≥4 mice of each cohort for each of the following parameters.  NOTE: To maintain consistency analyze crypts from the same location of each intestine (only use the proximal end).
2. Cellular parameters from standard H&E stained sections (unless otherwise stated): Crypt number, height, apoptosis and mitosis.
   1. Crypt number.
      1. Use a low power magnification (e.g. 4X or 10X) to manually count the number of crypts in contact with the basal layer.  Count ≥10 transverse proximal intestine sections from at least 4 mice.
   2. Crypt height.
      1. Use a high power magnification (e.g. 20X or 40X) to manually count the number of cells from the bottom of the crypt to the crypt/villus axis (Figure 3b).
   3. Apoptosis^22,23.
      1. Method 1: Use a high power magnification (e.g. 20X or 40X) to manually count the numbers of apoptotic cells in each crypt.  Apoptotic cells can be identified by cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies (Figure 3a).
      2. Method 2: Perform an IHC stain (section 7) for Caspase-3 using conditions described in Table 2.  Using a high power magnification (e.g. 20X or 40X) manually count the number of positive cells in each crypt to quantitate the cells in the execution phase of apoptosis.
   4. Mitosis.
      1. Use a high power magnification (e.g. 20X or 40X) to  manually count the numbers of mitotic cells in each crypt.  Mitotic cells contain condensed DNA material and are typically symmetric and well formed (Figure 3b).
   5. Proliferation.
      1. Perform an IHC (section 7) stain for Ki-67 using conditions described in Table 2.  Manually count positive cells to quantitate the proportion of crypt cells proliferating.