Figure 1.
Workflow for one step, room-temperature phosphoprotein preservation with subsequent paraffin embedding.
A novel tissue preservation solution, biomarker and tissue histology preservative (BHP), stabilizes phosphoproteins and cellular morphology at room temperature. The application combines the unique advantages of snap-freezing (immediate and excellent preservation of phosphoproteins, as well as high protein yield) and formalin fixation (preservation of tissue morphology and antigenicity) in one paraffin block.
Table 1.
Biomarker and Histology Preservative (BHP) composition.
Figure 2.
Protein yield from BHP-fixed tissue is equivalent to frozen tissue.
Comparison of extractable protein yield from paraffin embedded BHP-fixed and formalin-fixed mouse brain and liver tissue to matched snap-frozen samples. Even after seven days of fixation in BHP and using a short eight-minute extraction protocol the total protein yield is very high compared to snap-frozen tissue. (white bar = formalin fixed 1 day ±SEM; gray bar = formalin fixed 7 days ±SEM; black bar = BHP fixed 7 days ±SEM).
Figure 3.
Phosphoprotein and total protein levels are stable for one week in BHP fixative.
Six aliquots of human colon mucosa were either fixed for two or seven days in BHP and paraffin embedded. Reverse phase protein microarray analysis of 11 phosphoproteins, 10 total protein, and 2 cleaved protein modification endpoints revealed no significant difference in protein abundance due to fixation time (open circle = 2 day fixation, black square = 7 day fixation, Mann Whitney test, p>0.05).
Figure 4.
Evaluation of protein and phosphoprotein preservation by BHP fixation.
(A) Slight tissue-architectural differences between patients don't appear to affect fixation efficiency by BHP. The reverse phase protein microarray analysis of 19 endpoints in human colon mucosa from two different patients revealed an almost identical pattern of post-translationally modified and total protein abundances. (B) BHP fixation rapidly reduces tissue tyrosine phosphatase activity. Human breast tissue samples were directly lysed or fixed in BHP or formalin for 1, 5 and 15 minutes prior to lysis. Residual total tyrosine phosphatase activity was measured using an ELISA. After one minute of BHP fixation phosphatase activity was reduced to 18% of non-fixed levels and was completely absent by 15 minutes of fixation. In contrast, formalin fixation retained 71% phosphatase activity after 1 minute of fixation and 29% by 15 minutes of fixation. (n = 2, black circle-formalin; white square = BHP, dashed line non-fixed; ±SEM). (C) BHP fixation followed by paraffin embedding yields comparable levels of post-translationally modified and total protein in mouse liver tissue compared to snap-frozen samples for 16/17 endpoints. Mouse liver (n = 3, ±SEM) was either paraffin embedded after fixing in BHP for seven days or snap-frozen immediately and analyzed on a reverse phase protein microarray (open circle = snap frozen; black square = BHP 7 day fixation).
Figure 5.
BHP fixation preserves dynamic changes in phospho-EGFR Tyr1068 abundance post stimulation.
(A) Schematic of experimental setup. U266 cells were continuously stimulated with EGF for 30 minutes. Cell aliquots at 0, 10 and 30 minutes were removed and directly lysed or fixed in BHP or formalin prior to lysis. (B) After 10 minutes of fixation, BHP-fixed and formalin-fixed cells correctly maintain the expected pattern of phospho-EGFR Tyr1068 increase and decrease that is observed in non-fixed cells. (C) While BHP-fixed cells still maintain the expected phosphorylation pattern after two hours of fixation, this pattern is lost in formalin-fixed cells. (n = 3, black diamond = formalin; black square = BHP; open circle = non-fixed; ±SEM; error bars for the samples directly lysed after 30 minutes of EGF stimulation are very small and masked by the open circle symbol).
Figure 6.
Comparison of tissue morphology between paraffin blocks of BHP-fixed or formalin-fixed mouse and feline tissue.
BHP fixed or fomalin fixed matched tissue sections were stained with Hematoxylin and eosin (H&E). BHP fixation demonstrates equivalent preservation of cellular morphology with full cytoplasmic and membrane detail compared to formalin fixation. Nuclear structures (chromatin and nucleoli) are generally better preserved by BHP (20x magnification, inset 100x) (Continued in Figure 7) (For description of histological details see Text S1).
Figure 7.
Comparison of tissue morphology between paraffin blocks of BHP-fixed or formalin-fixed mouse tissue.
In addition to excellent preservation of cytoplasmic, membrane and nuclear detail, BHP-fixation eliminates the need for decalcification of bony tissue. Mouse tail, rib, and femur samples were fixed in BHP and paraffin embedded without decalcification prior to embedding. Bony structures remain intact and were able to be cut into sections (5 µm) using standard sectioning protocols (H&E stain, 20x magnification, inset 100x) (For description of histological details see Text S1).
Figure 8.
Human tissue fixed in BHP retains morphology equivalent to formalin.
Matched human tissue samples were fixed in either BHP or formalin and paraffin embedded. The same pattern as seen for mouse and feline tissues (see Figures 5 and 6) were observed for human tissue fixed in the one-step biomarker and histology preservative: equivalent preservation of cytoplasmic and membrane detail, with superior preservation of nuclear structures as compared to matched formalin fixed paraffin embedded tissue (H&E stain, 20x magnification, inset 100x) (For description of histological details see Text S1).
Table 2.
Independent evaluation of human colon mucosa by two pathologists.
Figure 9.
Nuclear size is maintained in tissue fixed with the novel biomarker and histology preservative.
The nuclear area of BHP-fixed or formalin-fixed and paraffin-embedded human colon mucosa, mouse brain purkinje cells, mouse liver hepatocytes and mouse pancrease islet of Langerhans cells was measured using the Arcturus XT platform (n = 50–100). No shrinkage in nuclear size was observed after BHP fixation. (white bar = formalin; black bar = formalin ±SEM).
Figure 10.
Biomarker and histology preservative shows equivalent or enhanced antigenicity by immunohistochemistry compared to FFPE.
To show that our new fixation chemistries maintains antigenicity for common diagnostic antigens, the effect of BHP fixation on non-phosphorylated protein antigenicity was compared in human tissue fixed in BHP or formalin and paraffin embedded in an international, multi-center study. At each institution, sections were stained side-by-side, using the same protocol for each fixative. Staining and embedding protocols varied between institutions and for different antigens. Antigenicity was stronger for most endpoints in BHP-fixed tissue, with the exception of CDX2 (SMA = smooth muscle actin, ERα = estrogen receptor alpha, PR = progesterone receptor, PAS = Periodic Acid Schiff stain, DCIS = ductal carcinoma in-situ, DLBCL = diffuse large B-cell lymphoma, LN met/col. canc. = lymph node metastasis from colon cancer) (For description of histological details see Text S1).
Figure 11.
BHP fixed tissues retain phosphoprotein antigenicity with immunhistochemistry.
(A) Effect of BHP fixation on phosphorylated protein antigenicity. Human and mouse tissues were fixed with BHP or formalin, followed by paraffin embedding. Sections were stained side-by-side, using the same protocol for each fixative. Staining intensity with phospho-specific antibodies was stronger after BHP fixation for every analyte tested. (B) Effect of long-term BHP fixation on protein antigenicity. Human colon was fixed for three months in BHP, followed by paraffin embedding. Specific and strong staining was observed for phospho-ERK Thr202/Tyr204 and cytokeratin 20, indicating retention of antigenicity for immunohistochemical studies (For description of histological details see Text S1).