Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Summary

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Abstract

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Introduction

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to disease or surgery^1,2. After trauma, the gut undergoes a morphometric and functional adaptive response, characterized by crypt cell proliferation and increased nutrient absorption³. This step is critical in recovery, yet poorly understood. Experimental studies of the intestinal adaptive response have focused on the changes occurring after small bowel resection in mice, rats, and pigs, but understanding the molecular mechanism behind the adaptive response in other kinds of injuries (e.g., chemical or bacterial) is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Experimentally, different approaches have been used to describe the complex molecular and cellular index of small intestinal pathology, including histopathological scoring and measuring the outcome of injury. Despite this, what is absent from the literature is a detailed description of how to perform the procedures that are needed to obtain reproducible data. When identifying factors involved in adaptation, such as gut hormones, an easy, low cost, and reproducible animal model is warranted and here we suggest using a model of chemotherapy-induced intestinal mucositis (CIM).

One of the simplest and very informative endpoints of both injury and adaptation is to measure the mass of the small intestine (SI). We know that a hallmark of mucositis is apoptosis of enterocytes, time-dependent villus atrophy and reduced mitosis. Therefore, examining intestinal morphology is highly relevant in preclinical models^4,5. In humans, a decline in plasma citrulline, a marker of functioning enterocytes, correlates with toxicity scores and inflammatory markers⁶ in addition to the absorptive capacity⁷, suggesting this amino acid is an excellent biomarker of mucositis. Citrulline can be measured in both mice and rats, and has shown excellent correlations with villus length⁸, crypt survival⁹, and radiation-induced mucositis¹⁰.

A major advantage of measuring plasma citrulline is the ability to collect repeated measurements from one animal. However, multiple blood sampling in mice is restricted to a total blood volume of 6 µL/g/week and requires general anaesthesia. This unfortunately also limits the use of citrulline measurements in mice. Furthermore, the measurement of citrulline requires high-performance liquid chromatography^11,12, which is costly and time-consuming. Recently, we showed that citrulline levels in mice correlate significantly with SI weight (p < 0.001) (unpublished data), making citrulline a direct measurement reflecting enterocyte mass. A limitation to the measurement of SI weight is the necessity for the mice to be sacrificed and thus no repeated measurements within the same mouse are possible. Still the method provides the possibility to perform a variety of other tissue analyses directed to the research question, and these facts can conceivably make up for the additional use of animals. We, therefore, suggest using SI weight as an easy, low-cost, and fast biomarker of injury and adaptation in mice. To ensure reproducibility and acceptable analytic variation, the intestines should be carefully removed from the animal, flushed with saline, emptied and dried before weighing. In this article, we show exactly how this procedure is performed.

Another hallmark of mucositis is the loss of the proliferating cells in the crypts and a compensatory hyperproliferation during the regenerative period³. The cellular marker Ki67 has been frequently used to determine fast proliferative cells by means of immunohistochemistry¹³. Even though Ki67 is a simple marker of proliferation, it has a tendency for imprecision as Ki67 is present during all active phases of the cell cycle (G1, S, G2, and M)¹⁴. Specific labelling is essential to detect replicating cells, which is why we suggest in situ incorporation of 5-bromo-2'-deoxyuridine (BrdU), a synthetic analogue of thymidine, as it is largely restricted to replicating cells in the S-phase¹⁵. BrdU is injected in the animals 150 minutes before sacrificing and cells can be subsequently detected with immunohistochemistry using BrdU specific antibodies. In this method article, we show exactly how to measure the area of BrdU immunopositive cells within a crypt using a free image software.

Morphologic and functional changes are often studied in 5-FU induced mucositis models, where the intestinal adaptation is assessed by villus height and crypt depth. During this study, we found that during the acute phase of mucositis, which is equal to the injury phase, proliferation measured by BrdU incorporation is not correlated with crypt depth. In contrast to this, crypt depth is significantly correlated with proliferation seen in the repair phase of mucositis, 3 to 5 days after induction. This suggests that the acute phase of mucositis is not measurable by crypt depth alone. We suggest that when using proliferation as an endpoint in the acute phase of mucositis mice, BrdU incorporation should preferably be used but when quantitating hyperproliferation in the later stage during the regenerative phase, crypt depth is a reasonable alternative to BrdU incorporation. The goal of this study was to describe this model in a way that it can be used by all researchers, both in the field of oncology but especially researchers not familiar with intestinal injury models.

The described model can be used to phenotype transgenic models according to the adaptive response using body weight, SI weight and crypt depth as endpoints. As an example, we show here how we used the model of 5-fluorouracil (5-FU) induced mucositis in a cellular knock out model with insufficient L-cell secretion¹⁶. Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are intestinal hormones co-secreted from the enteroendocrine L-cells in response to food intake^17,18. GLP-2 is recognized as an important factor for intestinal healing, the regulation of mucosal apoptosis and the improvement of the barrier function of the SI^19,20,21,22. Based on the literature, we hypothesized that endogenous hormones are essential for compensatory hyperproliferation occurring in the adaptive response after injury.

Protocol

All methods described were conducted in accordance with the guidelines of Danish legislation governing animal experimentation (1987). Studies were performed with the permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee.

NOTE: Female C57BL/6J mice (~20−25 g) were obtained and housed eight per cage in standard 12 h light, 12 h dark cycle with free access to water and standard chow. Animals were left to acclimatize for one week before experiments began.

1. Induction of mucositis using 5-fluorouracil

1. Obtain 5-fluorouracil (5-FU) in a 50 mg/mL solution.
2. Weigh and record the body weight of the mice. From the body weight, calculate the amount of 5-FU for injection (e.g., 400 mg/kg).
3. Prepare a 1 mL syringe connected to a 27 G x 30 mm needle and fill the syringe with the calculated amount of 5-FU for injection.
4. Restrain the mouse by the scruff method. To do this, grab the base of the tail with one hand and place it on a toe-gripping surface, such as a wire bar lid. While positioning the tail with one hand, hold the scruff of the neck with the other hand.
5. Firmly position the body of the mouse across one hand by extending the forefinger and thumb back as far as possible. Place the tail between the fingers of this same hand to secure the mouse.
6. While maintaining the mouse in a firm but gentle grip, expose the ventral side of the mouse and insert the needle into the intraperitoneal cavity on the lower right or left quadrant of the abdomen. Aspirate to ensure proper placement and inject the 400 mg/kg 5-FU.

2. Tissue collection

1. Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) diluted in saline solution (0.9% NaCl). Monitor the depth of anaesthesia by observing the pinch withdrawal reflex.
   NOTE: The anaesthesia is a non-recovery anaesthesia.
2. Record the weight of the anesthetized mouse.
3. Place the mouse in a supine position and perform a laparotomy to expose the abdominal cavity, followed by an incision of the chest cavity to introduce pneumothorax.
4. Using scissors, sacrifice the animal by cutting open the diaphragm.
5. Remove the small intestine. Cut superior to the pylorus, carefully retract the small intestine away from the carcass until the cecum is reached and make a cut just before the cecum.
6. Using forceps, gently clamp the proximal lumen shut. Using a 1 mL syringe with a 25 G needle attached, flush the small intestine with saline to remove the feces.
7. Gently remove the saline from the intestinal lumen with surgical instruments.
8. Place the small intestine on clean blotting paper to carefully remove excess saline.
9. Record the weight of the flushed tissue.
10. Calculate the flushed small intestinal weight as a percentage of body weight.

3. Small intestine histology

1. Tissue preparation
   1. In a fume hood, use scissors to cut 3 cm sections of the flushed intestinal tissue from each mouse from the duodenum, jejunum and ileum and fixate sections in 10% formalin for 24 h at room temperature.
      NOTE: The fixative volume should be 5−10 times of the tissue volume.
   2. Transfer the fixed tissue to a cutting board.
   3. Using a scalpel, trim the tissue to approximately 1 cm and transfer into an embedding cassette.
      NOTE: The cassette should be labelled with the sample identification (ID) in pencil.
   4. Submerge the cassette in 70% ethanol and store at 4 °C until further processing.
2. Tissue embedding in paraffin
   1. Dehydrate the tissue by placing in ascending alcohol solutions. Place the cassette in 70% ethanol for 1 h, followed by 80% ethanol for 1 h, 95% ethanol for 1 h and 100% ethanol for 1.5 h. Transfer the cassette from 100% ethanol to xylene for 1.5 h.
   2. Immerse the cassette with paraffin wax heated between 58−60 °C. Use forceps to position the tissue in a transverse orientation and leave the blocks to cool more than 24 h until solid.
3. Cutting tissue using a microtome
   1. Place block, tissue face down on ice for 5 min. Using a microtome, trim the paraffin blocks at a thickness of 10−30 µm to expose the tissue surface.
   2. Cut cross-sections of the tissue block between 3−5 µm, making transverse sections from the duodenum, the jejunum and the ileum.
   3. Place paraffin ribbon in a water bath set between 40−45 °C. Use forceps to separate the sections.
   4. Use microscope slides to pick up the sections from the water.
   5. Air dry the slides for 30 min before staining.
      NOTE: For storage over a longer period of time, store slides in a fridge.
4. Hematoxylin-eosin staining of tissue
   1. Place the microscope slides in a histology cradle. Deparaffinize the slides in a heating cabinet set at 60 °C for 60 min.
      NOTE: Slides directly from the fridge should be left to reach room temperature before placing them in a heating cabinet.
   2. In the fume hood, rehydrate the tissue in 3 changes of a clearing agent (Table of Materials), make 20 dips in the first clearing agent jar and then let them stand for 7−8 min in each of the two additional clearing agent jars. Transfer the slides to descending alcohol solutions, start with 3 changes of 99% ethanol, followed by 2 changes of 96% ethanol and 70% ethanol. Make a minimum of 20 dips in each jar. Transfer to running water and let the slides stand for 5 min.
   3. Immerse the slides in filtered Meyer’s hematoxylin for 1 min.
   4. Wash the samples in running tap water for 5 min.
   5. Immerse sections in the eosin stain for 1−2 min, then rinse in tap water.
   6. Dehydrate the tissue by placing in ascending alcohol solutions. Start with 70% ethanol, followed by 96% ethanol, 96% ethanol, and 4 times 99% ethanol. Make a minimum of 20 dips in each jar, and let the cradle stand in 99% ethanol until they are mounted.
   7. Mount coverslips by applying a small amount of the mounting medium to the surface of the slides. Put the coverslip on top of the mounting medium without creating bubbles under the coverslip. Let it dry for 24 h.
   8. Examine the tissue using a light microscope connected to a camera to obtain histological photos. Use a 10x objective to take snapshots until a full coverage of the tissue slide is reached.

4. Measurement of crypt depth and/or villus height

1. Download and install the analysis software (i.e., Zen Lite, Table of Materials).
2. Open the image in the software and connect to the camera. Take snapshots in camera mode with 20x objective.
3. In processing mode, open the snapshot.
4. To measure the crypt depth: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated crypt and start the line at the bottom of a villus and finish at the bottom of the crypt. Repeat this action for 20 well-orientated crypts (Supplementary Figure 1).
5. To measure the villus height: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated villus and start the line at the end of the luminal projection and finish at the start of the crypt. Repeat this action for 20 well-orientated villi (Supplementary Figure 1).
6. Switch from 2D view to the Measure view to display the measurements.
7. Export the measurements and calculate the average of the crypt depth and villus height.

5. BrdU quantification (proliferation) by immunohistochemistry

1. Injection of the BrdU solution
   1. Weigh and record the body weight of the mice.
   2. In a fume hood, prepare a representative amount of 0.5% w/v bromodeoxyuridine (BrdU) solution in phosphate buffered saline (PBS).
   3. In the fume hood, inject 50 mg/kg of BrdU by intraperitoneal injection.
      NOTE: To ensure that each mouse is euthanized at exactly 150 min post BrdU injection, inject each individual mouse in succession with 10−20 min interval in between in order to properly perform the tissue collection.
   4. Record the time of injection.
   5. Wait 150 min, then anesthetize mice and collect the tissue as described in steps 2.1−2.7. Perform BrdU immunohistochemistry as described in section 5.2.
2. BrdU immunohistochemistry
   1. Prepare the tissue as described in steps 3.1.1−3.3.4.
   2. For antigen retrieval, place a slide cradle with the sections in a glass histology jar and fill it with the EDTA buffer (pH 9) in a microwave oven for 1 min at 750 W followed by 9 min at 350 W. Repeat cycle.
      NOTE: Ensure that the tissue does not dry out, which might require adding more buffer to the jars between heating.
   3. Wash sections 2−3 times in tris-buffered saline and polysorbate 20 (TBS-T) buffer for 3 min each.
   4. Apply 5 drops of peroxide block (Table of Materials) for 10−15 min at room temperature to block endogenous peroxidase activity. Wash sections 2−3 times in TBS-T buffer for 3 min each.
   5. Apply 5 drops of rodent block buffer (Table of Materials) for 30 min at room temperature, to block non-specific binding. Wash sections 2−3 times in TBS-T buffer for 3 min each.
   6. Incubate the sections with a monoclonal mouse anti-BrdU antibody diluted 1:500 for 1 h at room temperature. Wash sections 2−3 times in TBS-T buffer for 3 min each.
   7. Visualize immunopositivity using 3,3’-diaminobenzidine (DAB) by applying 5 drops of horseradish peroxidase to each slide and incubate for 15 min at room temperature. Transfer the sections to a fume hood, add 150 µL of DAB and incubate for 5 min.
      NOTE: Always wear gloves when handling DAB and dispose of waste appropriately.
   8. Rinse sections in deionized water.
   9. Dehydrate the tissue and mount a coverslip as described in steps 3.4.6 and 3.4.7.
3. Visualization of the immunoreactions
   1. Visualize the tissue sample with a light microscope connected to a camera.
   2. Use the analysis software to obtain microscope images using a 20x objective of all sections and save files in the .JPG format.
   3. Take a snapshot of a stage micrometer using a 20x objective, which will be used as a calibration tool in ImageJ software.
4. Measuring the area of BrdU immunoreactive cells
   1. Install ImageJ software (Table of Materials).
   2. Assigning scale to an image in ImageJ: Open the stage micrometer image using the ImageJ File | Open menu command. Select the straight-line selection tool.
   3. Select the straight tool and draw a straight line on the micrometer image to define a known distance.
   4. Select Set Scale under the Analyse menu.
   5. Enter a value in the Known Distance box and define the unit of length in the Unit of Length box. Select Global so that this calibration applies to all images that are opened in this ImageJ session.
   6. Open an image of interest by using the ImageJ File | Open menu command to measure the area of the BrdU immunoreactive cells per crypt.
   7. Set the color graphics to 8-bit under the Image/Type menu.
   8. Increase the contrast of the image under Process/Enhance contrast and set the saturated pixels to 0.4%.
   9. Apply a threshold to the image to segment the immunopositivity. Select Image/Adjust under menu, then threshold and select the color Red (dark area shown in red). Choose a threshold of around 10−20% by moving the threshold bar.
      NOTE: Choose a threshold that includes all the BrdU positive cells with as little background as possible. The chosen percentage should apply to all sections.
   10. Choose a well-orientated crypt, i.e., a complete crypt from intact tissue, and select the free hand selection tool to mark the area around it.
   11. To measure the threshold area, select the Analyse tab, followed by Analyse particles. In the Analyse particle window, set Size to 20-infinity, click the box pixel units, set Circularity to 0.00−1.00, and set Show to Outlines. Click boxes Display Results, Summarize, and Include holes.
       NOTE: The values for BrdU positive cells are automatically generated in a result window, showing the individual measurements for each BrdU positive cell. Furthermore, a summary window is automatically generated, showing the number of counts, total area, average size and percentage of area with BrdU positive cells.
   12. Repeat steps 5.4.10 and 5.4.11 to measure a new crypt. The results of all measured crypts will be displayed in the Summary window.

Representative Results

In the first experiment, we induced mucositis in mice at day 0 and sacrificed a group of mice each day for 5 consecutive days. When measuring the SI weight, we found that this parameter decreased from day 2 until day 4 suggesting a loss in the enterocyte mass. We also found that at day 5, the SI weight was not significantly different from day 0 (untreated mice) (Figure 1). The proliferation measured by the incorporation of BrdU was almost abolished at day 1 and day 2, but at day 4 and day 5 there were approximately four-fold and five-fold increases in proliferation, respectively (Figure 2). This hyperproliferation was also illustrated when measuring the crypt depth (Figure 3). What is not illustrated by measuring crypt depth is the loss in proliferating cells at day 1 and day 2, where crypt depth was reduced by approximately 13% but was not significantly different from the healthy mice. We could show that, in the regenerative phase of mucositis there was a strong correlation between BrdU incorporation and crypt depth, but this did not count for the acute phase indicating that crypt depth as an endpoint might not be suitable in the acute phase of mucositis (Figure 4).

In the second study, we examined mucostis in our transgenic mouse model with insufficient L cell secretion. Mice with deficient GLP-1 and GLP-2 showed a severe loss of body weight (BW) and a decrease in SI weight in the recovery phase, which was highly significant compared to the wild type (WT) 5-FU mice (p < 0.01) (Figure 5A,B). Furthermore, the transgenic mice failed to show compensatory hyperproliferation; crypts were significantly shorter than in both WT mice and healthy controls. Contrary to this the WT mice showed an increase in crypt depth as a sign of hyperproliferation (Figure 5C).

Figure 1: Small intestinal weight. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and the intestinal weight was measured as described. Results are shown as mean ± standard error of the mean (SEM). n = 13. This figure has been modified from Hytting-Andreasen et al.¹⁶. Please click here to view a larger version of this figure.

Figure 2: BrdU immunopositive cells per crypt for the duodenum, jejunum, and ileum of the SI. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and BrdU incorporation was quantified by immunohistochemistry as described. Results are shown as mean ± SEM. n = 13. *p < 0.05, ***p < 0.001 compared to day 0 (analysis of variance [ANOVA] followed by Dunnett multiple comparison test). ***p < 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.¹⁶. Please click here to view a larger version of this figure.

Figure 3: Measurement of crypt depth. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and crypt depth was measured as described. Results are shown as mean ± SEM. n = 13. *p < 0.05, ***p < 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.¹⁶. Please click here to view a larger version of this figure.

Figure 4: Correlation of crypt depth and BrdU. Crypt depth (in duodenum, jejunum, and ileum) is correlated to BrdU incorporation at each day of sacrifice using two-tailed Pearson correlation test. Please click here to view a larger version of this figure.

Figure 5: GLP-1 and GLP-2 deficient mice fail to regenerate after acute mucositis. (A) Change in BW (%), (B) SI weight (g), and (C) crypt depth in ileum (µm). Results are shown as mean ± SEM. Tg = transgenic mice; WT = wild type mice; 5-FU = 5-fluorouracil. n = 4−8. *p < 0.05, **p < 0.01, ***p < 0.001 compared to healthy control (WT saline), a = p < 0.05, aa = p < 0.01, aaa = p < 0.001 compared to WT 5-FU (two-way ANOVA followed by Bonferroni multiple comparison test [BW] or ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.¹⁶. Please click here to view a larger version of this figure.

Supplementary Figure 1: Small intestinal tissue before the induction of mucositis, during the acute phase and the recovery phase of mucositis. (A) Haemotoxylin and eosin (H&E) staining and (D) BrdU staining in untreated mice. The black dotted line in panel A exemplifies a well-orientated crypt, whereas the dotted green line demonstrates a well-orientated villus. (B) H&E staining and (E) BrdU staining during the acute phase of mucositis. (C) H&E staining and (F) BrdU staining during the recovery phase after the induction of mucositis. Scale bar = 100 μm. Please click here to download this file.

Discussion

Here, we demonstrate a widely accessible method to study SI injury and regeneration in a mouse model. A wide variety of preclinical animal models of intestinal injury exist, but it is vital we understand that each model is unique and that the endpoints must be appropriate to answer the research question. This model is excellent to study adaptive response to injury, but the endpoints should be modified when using the model as a pre-clinical model of mucositis. However, translation from animal models to patients is challenging²³. Our suggested endpoints of SI weight and proliferation should be limited to the study of adaptive response only. The study of endogenous factors often requires the use of transgenic mice, and even if small bowel resection is possible in mice¹, this model could be an alternative to avoid post-operative mortality. When applying this model to transgenic mice, it is important to watch the mice carefully and monitor their weight every day. During this study, some of the mice experienced a weight loss of up to 30%, which is quite substantial. To avoid high mortality in sensitive phenotypes, we suggest performing pilot studies in transgenic mice, since dose adjusting might be necessary.

A critical step within the described method is the removal and weighing of the SI. It is important that the removal and handling be performed in the same manner and by the same researcher each time to avoid large inter-assay variations.

The consistency of crypt and villus selection is important to avoid variance and bias when measuring crypt depth and villus height. When embedding the tissue in paraffin, the intestines are positioned in an upright position to make transverse cuts, thus increasing the possibility for intact villi and crypts. After cutting of the tissue, a well-orientated crypt and/or villus is selected. Selection is based on the full visualization of the whole crypt and villus in the same plane and the presence of clear borders of cells within the crypt and villus. A limitation to this method is the somewhat subjective approach when selecting a well-orientated crypt since the selection of a well-orientated crypt and/or villi is made after cutting the tissue. A previous study²⁴ has presented an alternative method to overcome this limitation, where they use microdissection. In this method, villi and crypts are selected while observing under a microscope, prior to the tissue being cut, thus making it possible to ensure that an intact crypt and villi are being dissected from the tissue.

In contrast to previous methods used to quantity BrdU positive cells^25,26, this protocol describes the area of BrdU positive cells per crypt, which provides a fast way to quantitate proliferative cells within each crypt. This technique, however, may be somewhat restrictive since it requires a more profound knowledge of the software suggested for the measurement of this technique. A future application of this protocol could be to create a more automatic generated method to quantify and measure the BrdU positive cells.

Materials

Name	Company	Catalog Number	Comments
5-Fluorouracil	Hospira Nordic AB, Sweden	137853
Ketaminol®Vet	Merck, New Jersey, USA	511485
Rompun®Vet Xylazine	Rompunvet, Bayer, Leverkusen, Germany.	148999
10% nautral formalin buffer	Cell Path Ltd, Powys, United Kingdom	BAF-5000-08A
HistoClear	National Diagnostics, United Kingdom	HS-200
Pertex	HistoLab®, Sweden	840
BrdU	Sigma-Aldrich, Germany.	B5002
Tris/EDTA pH 9 buffer	Thermofisher scientific, Denmark	TA-125-PM4X
Peroxide Block	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
Rodent Block buffer	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
Monoclonal mouse anti-BrdU antibody	Thermofisher Scientific, Denmark.	MA1-81890
Lab Vision Antibody Diluent OP Quanto	Thermofisher Scientific, Denmark.	TA-125-ADQ
Horseradish peroxidase	Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark	TL-060-QHDM
DAB Quanto Substrate	DAB Substrate Kit, Thermofisher Scientific, Denmark	TA-125-QHDX
DAB Quanto Chromogen	DAB Substrate Kit, Thermofisher Scientific, Denmark	TA-125-QHDX
Zen Lite Software (Blue edition)	Carl Zeiss A/S		https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html
ImageJ Software	LOCI, University of Wisconsin		https://imagej.nih.gov/ij/