Name: tissue preparation and immunostaining of mouse craniofacial tissues and undecalcified bone
Uploaded: 2019-05-10T13:00:00
Description: Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone at JoVE.com

This work was supported by the National Institutes of Health (R01DE020843 to Y.M.), the International FOP Association (Y.M.), and a grant-in-aid from the National Natural Science Foundation of China (31500788 to J.Y.).

All mouse experiments were carried out in accordance with University of Michigan guidelines covering the humane care and use of animals in research. All animal procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan (Protocol #PRO00007715).
1. Tissue Preparation
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	<li>Preparation of embryonic tissues 
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		<li>Prepare one 10 cm dish and several 3.5 cm dishes containing phosphate buffered sali...

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Here we provide a detailed protocol for preparation of mouse head and undecalcified bone tissues, and cryosectioning for immunostaining of cell proliferation, cell death, and BMP signaling markers. We also detail the strategy for obtaining quantitative data from immunofluorescent images. Those methods can also be applicable to other tissues with appropriate modifications.
Conditions for tissue preparation vary by the size and type of tissues. The fixation and cryoprotection time usually need s...

The face is a key part of human identity, and is composed of several different types of tissues, such as epithelium, muscle, bone, cartilage, tooth. Those tissues are derived from all three germ layers: ectoderm, endoderm, and mesoderm1,2. For proper patterning and development of craniofacial tissues, cell proliferation, death and differentiation need to be highly coordinated and regulated by specific signaling pathways, such as Wnt, Fgf, Hh and Bmp pathways3,4,5. Defects in proliferation, survival or differentiation of cells will lead to craniofacial malformations, which are among the most frequently occurring congenital birth defects. Transgenic mice are useful tools to study mechanisms of craniofacial morphogenesis and pathogenesis1,2,3,4,5. Understanding the changes in craniofacial structures during development and pathogenesis will help to clarify key developmental principles as well as the mechanisms of craniofacial malformations1,2,3,4,5.
The staining of whole mount or sectioned tissues with specific antibodies is an invaluable technique for determining spatial distribution of proteins of interest 6. Formally, tissue immunostaining can rely either upon immunohistochemistry (IHC) or immunofluorescence (IF). Compared with the opaque reaction product generated with a chromogenic substrate such as 3,3&#8217;-Diaminobenzidine (DAB) by IHC, IF involves the use of fluorescent conjugates visible by fluorescence microscopy. Therefore, IF may clearly differentiate positive cells from background noise, and allows images to be quantitatively analyzed and enhanced in a straightforward fashion by software such as ImageJ and Adobe Photoshop7,8. The whole mount staining approach works on small blocks of tissue (less than 5 mm thick), which can provide three-dimensional information about the location of proteins/antigens without the need for reconstruction from sections9,10. However, compared with tissue sections, whole mount immunostaining is time consuming and requires large volumes of antibody solutions. Not all antibodies are compatible with the basic whole mount approach. In addition, the incomplete penetration of antibodies will result in uneven staining or false negative staining. Here we will focus on the immunofluorescence detection of proteins/antigens on sectioned tissues. For hard tissues (eg, head, tooth, long bone), calcium deposition during development/pathogenesis makes the sample difficult to section and easily rinsed off during immunostaining treatment11,12. Most of the currently available protocols decalcify hard tissues before embedding to make sectioning easier, which is time consuming and can destroy morphology and antigens of samples if handled improperly11,12. To overcome the issues, we optimized an approach for cryosectioning of hard tissues without decalcification, leading to improved visualization of their morphology and distribution of signaling proteins.
The protocol described here is being used to determine morphometric and histological changes in the craniofacial tissues of BMP transgenic mice. Specifically, the protocol includes (1) harvesting and dissecting head tissues, (2) section and immunostaining of experimental markers (Ki67, pSmad1/5/9) along with TUNEL staining, (3) imaging the sections using fluorescence microscope, and finally (4) analyzing and quantifying the results. The protocol to prepare and cryosection hard tissues without decalcification is also described13. Those methods are optimized for craniofacial tissues. They are also applicable to other tissues from various ages of samples with appropriate modifications.

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			<td height="21" style="height:21px;width:296px;">Adhesive tape</td>
			<td style="width:171px;">Leica</td>
			<td style="width:119px;">#39475214</td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:296px;">Alexa fluor 488-goat anti-Rabbit secondary antibody</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">A-11034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Antifade Mountant with DAPI</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">P36931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Coverslips</td>
			<td style="width:171px;">Fisher Brand</td>
			<td style="width:119px;">12-545-E</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Cryostat</td>
			<td style="width:171px;">Leica</td>
			<td style="width:119px;">CM1850</td>
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			<td height="21" style="height:21px;width:296px;">EDTA</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Fluorescence microscope</td>
			<td style="width:171px;">Olympus</td>
			<td style="width:119px;">BX51</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Gelatin</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">In Situ&#160;Cell Death Detection Kit</td>
			<td style="width:171px;">Millipore</td>
			<td style="width:119px;">S7165</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Microscope slides</td>
			<td style="width:171px;">Fisher Brand</td>
			<td style="width:119px;">12-550-15</td>
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			<td height="21" style="height:21px;width:296px;">OCT Compound</td>
			<td style="width:171px;">Fisher Healthcare</td>
			<td style="width:119px;">23-730-571</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Paraformaldehyde (PFA)</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">P6148&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Phosphate buffered saline (PBS)</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">P4417</td>
			<td></td>
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			<td height="21" style="height:21px;width:296px;">Polyethylene glycol&#160;tert-octylphenyl ether</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td style="width:167px;">Triton X-100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Proteinase K</td>
			<td style="width:171px;">Invitrogen</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Rabbit anti-Ki67 antibody</td>
			<td style="width:171px;">Cell Signaling Technology</td>
			<td style="width:119px;">9129</td>
			<td>Lot#:3; RRID:AB_2687446</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Rabbit anti-pSmad1/5/9 antibody</td>
			<td style="width:171px;">Cell Signaling Technology</td>
			<td style="width:119px;">13820</td>
			<td>Lot#:3; RRID:AB_2493181</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sodium citrate</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">1613859</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Sucrose</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Tris</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:119px;">10708976001</td>
			<td></td>
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tissue preparation and immunostaining of mouse craniofacial tissues and undecalcified bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Embryonic craniofacial tissue sections 
Following the above steps, heads were dissected from control (P0-Cre) or mutant (constitutively activated Bmpr1a in neural crest cells, P0-Cre; caBmpr1a) embryos at embryonic day (E) 16.5 or 18.5. After fixing in 4% PFA for 4 h, samples were embedded in OCT and cryosectioned coronally. Resulted sections were immunostained with antibodies against pSmad1/5/9 (downstream BMP signaling factors) or Ki67 (a cell pro...

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone at JoVE.com

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Here, we present our simple protocol for immunofluorescence detection on proteins on sectioned materials. This method does not require antigen retrieval. We will use mouse heads, including undecalcified hard tissues.The main advantage of this technique is to skip antigen retrieval and decalcification of hard tissues. Those lead to a good quality of immunostaining results. This method also saves both time and labor.This method is also applicable to other tissues with appropriate modifications. To make nice sections from undecalcified tissues, dissect your target tissue further to trim surrounding tissues, and if necessary treat the samples with 10%EDTA at four degrees Celsius for two days before cryoprotection. Visual demonstration provides a detailed and a clear explanation of the procedure of this method.To begin, prepare one 10-centimeter and several 3.5-centimeter dishes containing PBS and one 12-well culture plate containing two milliliters of 4%PFA in PBS in each well for each pregnant mouse, and place them all on ice. To dissect embryos from pregnant mice, grab the skin below the center of the belly of a euthanized mouse with forceps, cut through the skin only, and then gently pull at the skin to separate it from the underlying abdominal muscle wall. Following the same line of the skin incision, cut into the abdominal cavity.Remove the uterus containing a string of embryos. Then, remove the embryos by gently cutting away the uterine wall, which will remove the extraembryonic tissues such as the yolk sac and amnion. Cut and isolate the head from each embryo, and with forceps transfer each head into a separate well of a 12-well plate containing 4%PFA.Incubate at four degrees Celsius for four hours to fix. Rinse the heads in PBS at four degrees Celsius with gentle shaking for 12 hours. To cryoprotect the heads, transfer them using forceps into a new 12-well plate containing two milliliters of 30%sucrose in PBS.Incubate with gentle agitation at four degrees Celsius until the heads sink to the bottom of the dish. To embed the cryoprotected heads, transfer them into a mold containing Optimal Cutting Temperature Compound, and equilibrate for several minutes. Using forceps, adjust the location and direction of the samples so that the trimmed side of the samples faces the bottom of the embedding mold.Then, place the mold on dry ice to freeze, and store in a plastic bag at minus 80 degrees Celsius until ready for cryosectioning. For postnatal tissue, after removing the skin and adipose tissues of a euthanized, three-weeks to three-months-old mouse, cut and isolate the head and remove the mandible. Then, fix and cryoprotect the head as done with embryo heads.Embed in 8%gelatin in a similar manner as embryo heads in OCT, and keep the Cryomolds in a plastic bag at minus 80 degrees Celsius until cryosectioning. Set the appropriate cryostat temperature. Place the samples in the cryostat chamber for about 30 minutes to equilibrate to the cryostat temperature.Remove the block with the sample from the Cryomold, and mount it with an OCT drop onto the specimen chuck and freeze it, making sure to keep the trimmed side of the sample furthest from the chuck and facing the operator. Load the block-mounted chuck onto the cryostat object holder. Adjust the blade holder so that the blade angle is three to five degrees relative to the sample.Collect 10-micrometer sections onto coated microscope slides. Leave the sections at room temperature until completely dry, and then store them at minus 80 degrees Celsius. To begin with the staining, take out slides from minus 80 degrees Celsius, and keep them at room temperature for one hour to air-dry the sections.Rinse the slides in 0.1%PBST three times for five minutes each to wash out OCT and permeabilize the sections. Add 200 microliters of blocking solution to each slide, and incubate at room temperature for 30 minutes. Then, remove the blocking solution without rinsing the slide.Add 100 microliters of primary antibody diluted in blocking solution to each slide, and incubate overnight at four degrees Celsius. Rinse the slides with PBS three times for 10 minutes each at room temperature. Add 100 microliters of secondary antibody diluted in blocking solution, and incubate for one hour at room temperature, protected from light.Rinse in PBS three times for 10 minutes each at room temperature, still protected from light. To mount the slides, add two drops of anti-fade medium with DAPI on each slide, cover with a coverslip, and store at four degrees Celsius until ready to image. Frontal bones of control embryos were positive for pSmad1/5/9, downstream BMP signaling factors, and Ki67, a cell proliferation marker.In neural crest-specific, constitutively activated BMPR1A mutant embryos, however, pSmad1/5/9 levels were increased while Ki67 levels were decreased. Furthermore, more apoptotic cells were observed in the frontal bones of mutant embryos than those of control embryos. Coronal cryosections of gelatin-embedded heads clearly show GFP signal and RFP signal from a tdTomato cassette in the nasal bone and nasal tissues, demonstrating that gelatin does not interfere with fluorescent signals.When these sections were immunostained for SOX9 or Osterix and E11/Podoplanin, good-quality sections were obtained from most of the three-week hard tissues. On the other hand, with three-month-old samples, good-quality sections were only obtained in some of the hard tissues, including the trabecular compartments of the femur, nasal tissues, and the skull, including the nasal-premaxilla suture and surrounding bones of the head. SOX9-positive cells were detected specifically in the chondrocytes of the growth plate and the joint from the femur and the nasal septum.In the three-week-old incisor, SOX9 was detected in the mesenchymal cells. Osterix and E11 double staining results show that Osterix was detected in osteoblasts, while E11 was detected in osteocytes of bones from the femur and the head. In the three-week incisor, Osterix was positive in odontoblasts, while E11 was positive in follicle mesenchymal cells.Please do not overfix samples with 4%PFA. For sectioning of undecalcified hard tissues in gelatin, best temperature is minus 25 degrees Celsius and lower. Following this procedure, levels of fluorescence can be quantified as described in our protocol.Those measurements will provide informative data to show the difference of the relative level of protein between different groups. After its development, this technique paved the way for double or triple staining to get co-expression patterns of different proteins. 4%PFA is hazardous.It may cause skin allergies, serious eye damage, organ damages, or cancer. Please use personal protective equipment, and wash skin thoroughly after handling.

tissue-preparation-immunostaining-mouse-craniofacial-tissues

Research

JoVE Journal

Developmental Biology

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...