Whole-mount in situ hybridization: minimizing the folding problem of thin-sheet tissue-like crayfish haematopoietic tissue

in Crustaceana
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Crayfish haematopoietic tissue (HPT) has a thin-sheet-like structure with a thickness of 100-160 μm and a width of approximately 1-2 cm. This structure makes HPT extremely easy to fold after removal from the animal. Therefore, it is difficult to handle the tissue without folding when processing for sectioning and histological study. The degree of tissue folding reflects the size of the tissue sections obtained, how complicated it is to interpret the location of each tissue section, and the accuracy of the interpretation of the location of a specific transcript. To facilitate the interpretation of a specific transcript location in the HPT, we optimized a whole-mount in situ hybridization technique to minimize tissue folding. This optimized protocol effectively reduced the tissue folding. Therefore, the location of a specific transcript in the HPT was easily and accurately defined. This protocol will be useful for whole-mount staining of other tissues with similar structure.

Whole-mount in situ hybridization: minimizing the folding problem of thin-sheet tissue-like crayfish haematopoietic tissue

in Crustaceana

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References

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Figures

  • View in gallery

    Structure of the crayfish haematopoietic tissue (HPT). The HPT is approximately 100-160 μm thick and at its widest point has a width of approximately 1-2 cm. The HPT is divided into two symmetrical sides by the ophthalmic artery. Each side of the tissue and the ophthalmic artery are connected at the anterior proliferation centre (APC), which is located at the most anterior part of the HPT.

  • View in gallery

    Cell strainers as tissue supports and carriers. Haematopoietic tissue is placed on a cell strainer (yellow) containing nylon mesh with 100-μm pore size, which is placed in a 6-well plate (A). The tissue can be easily transferred to different buffers, without being directly touched, using clean forceps to pick up the cell strainer (B).

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    HPT shrinkage and folding. A, Image of one half of HPT that was not subjected to hybridization at 60°C; B, image of one half of HPT which shrank after being incubated at 60°C when compared to the tissue in (A); C, the HPT was subjected to the whole-mount in situ hybridization protocol without adding any probe to the hybridization buffer and the hybridization was performed at 60°C. The 6-well plate was placed on shaker (low speed) during all washing steps, and the tissue was not covered with parafilm during prehybridization and hybridization steps. The tissue extensively folded during the process, and it was not possible to unfold the tissue. Therefore, the HPT lost its original structure at the end of the process; D-E, the HPTs were processed using 300 ng of sense RNA probe for CHF (D) and 300 ng of antisense RNA probe for CHF (E). The hybridization was performed at 58°C. With this amount of probe, the sense probe exhibited high background staining. Therefore, the amount of the probe was reduced to 100 ng for subsequent staining. For the staining of these two tissues, shaking was not performed during washing steps, and the tissues were covered with parafilm during the prehybridization and hybridization steps. Covering the tissue with parafilm, reducing hybridization temperature, and incubating tissues without shaking effectively minimized the tissue-folding problem. HPTs remained in their original structure until the end of the process. Scale bars are 1 mm.

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    A, Intracellular staining observed in the HPT incubated with antisense probes indicates sufficient tissue permeabilization. The HPTs were subjected to the whole-mount in situ hybridization protocol. Tissues were treated with proteinase K for 30 min before incubating in hybridization buffer without probe (B) which served as negative control for the staining protocol, or with CHF antisense probe (CHF-AS) (C), CHF sense probe (CHF-S) (D), Pl_PVR1 antisense probe (Pl_PVR1-AS) (E), or Pl_PVR1 sense probe (Pl_PVR1-S) (F). Cells with positive intracellular staining can be seen in (C) and (E). The black square in (A) indicates the area where the images in (B)-(F) were captured.

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    Staining results in the anterior part and the edge of HPT. A-C, Staining of CHF (crustacean haematopoietic factor) transcript in the anterior proliferation center (APC). The black square in the inset indicates the area where the image in (A) was captured. The black and red squares in (A) indicate the areas where the images in (B) and (C) were captured at higher magnification, respectively. White dashed lines in (B) and (C) indicate lobules that contain cells with CHF expression. Arrows in (C) indicate cells with CHF expression. D-F, Staining of CHF transcript in anterior part of HPT close to ophthalmic artery. The black square in the inset indicates the area where the images in (D) and (F) were captured. The black square in (D) indicates the area where the image in (E) was captured at higher magnification. The staining using sense probe gave low background staining (F). White and yellow dashed lines in (E) indicate lobules containing cells with and without CHF expression, respectively. Arrows in (E) indicate cells outside lobules with high CHF expression. G-I, Staining of the CHF transcript in one side of anterior part of the tissue. The black square in the inset indicates the area where the image in (G) was captured. The black square in (G) indicates the area where the image in (H) was captured at higher magnification. Cells at the edges have low or no CHF expression, whereas many cells in the centre exhibit strong positive staining (bordered by white dashed line in (H)). The staining using sense probe gave low background staining (I). J-L, Staining of CHF transcript at the edge of the tissue. The black square in the inset indicates the area where the image in (J) was captured. The black and red squares in (J) indicate the areas where the images in (K) and (L) were captured at higher magnification, respectively. Cells at the edges of the tissue have low or no CHF expression, whereas cells with high CHF expression localize toward the centre of the tissue (J). White dashed lines in (K) and (L) indicate lobule containing cells with high CHF expression. Yellow dashed lines in (K) and (L) indicate lobule containing cells with low CHF expression.

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    Staining results in the middle and the posterior part of HPT. A-F, Staining of CHF (crustacean haematopoietic factor) transcript in the middle area of the tissue. The black squares in the insets of (A), (C) and (D) indicate the areas where the images in (A), (C) and (D) were captured. The black squares in (A) and (D) indicate the areas where the images in (B), (E) and (F) were captured at higher magnification. A high density of cells with high CHF expression was observed in this area. Arrows in (B) indicate cells outside lobules with high CHF expression. Yellow dashed lines in (B), (E) and (F) indicate lobules containing cells with low CHF expression. White dashed lines in (E) and (F) indicate lobules containing cells with high CHF expression. Black dashed lines in (F) indicate lobule with no CHF expression. Staining using sense probe gave low background staining (C). G-I: CHF transcript staining in the posterior of the tissue. The black square in the inset indicates the area where the image in (G) was captured. The black square in (G) indicates the area where the image in (H) was captured at higher magnification. Cells at the edges have low or no CHF expression, whereas the cells with higher CHF expression localize toward the centre of the tissue. Yellow dashed lines in (H) indicate a lobule containing cells with low CHF expression. White dashed lines in (H) indicate lobules containing cells with high CHF expression. Staining using sense probe gave low background staining (I).

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