SDS-PAGE gel of moa extractions.

<p>Arrowheads indicate faint bands visible through the smearing.</p

Localization of antibody antigen (ab—ag) complexes <i>in situ</i> on feathers exposed to varied conditions.

<p>(A-B) Control, (C-D) 60°C, and (E-F) 350°C feathers. White barbs (A and C) are compared with brown barbs (B and D) for both the control and 60°C conditions. There are no noticeable differences in strength of binding between the pigmented and non-pigmented barbs in either condition. Antibodies bind with greater avidity in the feathers treated at 60°C, consistent with what we have observed in samples from other experiments that are partially degraded. (E) Background signal in the 350°C condition is weak and diffuse but binding is greater than the secondary antiserum only negative control (F) and localized to the keratinous ‘struts’ within the pith.</p

Summary of Rabilloud method, Craig and Collins method, Embery method, and Schweitzer method.

<p>*Dialysis and lyophilization is abbreviated D/L. Precip represents chloroform∶methanol∶water precipitation performed on half of the supernatant. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p

Barb fragment from 350°C feather.

<p>(A) Barb ramus retaining barbule protrusions (arrows) allows definitive identification as a barb fragment. Only the most proximal parts of the barbules can be observed where they branch from the ramus. (B) Transmitted light image of a 200nm thin section of a barb from the 350°C treated feather (similar to A) shows presence of external cortex (arrowhead) and inner pith observed as honey-comb texture.</p

Supplemental Methods and Figures from Microscopic and immunohistochemical analyses of the claw of the nesting dinosaur, <i>Citipati osmolskae</i>

Supplemental Methods and Figures for Moyer et al 201

TEM images of feathers from varied conditions.

<p>(A-B) Represent unpigmented barbs from the control feather and 60°C feather respectively. (C- D) Brown barb and barbule from control feather. (C) Represents barb where cortex (arrow) and inner pith are visible. Note melanosomes (arrowhead) are sparse but present only in barb cortex. (D) Barbule with melanosomes (arrow) from control feather. (E) 60°C brown barb with barbule extending from the left side. Melanosomes are concentrated in the barbule and appear partially degraded as indicated by the less dense (arrowhead) and even ‘hollow’ (arrow) centers observed in the inset. (F) Pith from a rachis taken from the 60°C condition, embedded and sectioned separately (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157699#sec005" target="_blank">Methods</a>). Note: Whether pith derives from the rachis (F) or barb (C) is impossible to determine at this level of magnification.</p

Localization of antibody antigen (ab—ag) complexes <i>in situ</i> on <i>Shuvuuia deserti</i> (IGM 100/977) filament.

<p>(A) Shows positive binding of the anti-chicken feather antiserum to the fossil tissue as indicated by the green fluorescent signal. (B) Demonstrates that the binding in A) is localized specifically to the tissue. (C and D) Secondary antiserum only control is negative and shows that the positive signal in A) is not due to spurious antibody binding.</p

TEM images of 350°C feather fragments.

<p>(A-B) Unidentifiable piece of 350°C feather at lower and higher magnification, respectively. The honey-comb structure observed in (A) indicates it is the pith of either a barb or rachis. (C-D) Feather fragment positively identified as barb. (C) External cortex (arrow) and internal pith are observed. (D) At higher magnification no electron dense microbodies consistent with melanosomes are observed in the cortex.</p

Summary of Franzén and Heinegård methods.

<p>*Dialysis and lyophilization is abbreviated D/L. Precip represents chloroform∶methanol∶water precipitation performed on half of the supernatant. Volumes correspond to the number of milliliters of buffer multiplied by the grams of bone powder.</p

Moa enzyme-linked immunosorbent assay results showing means plus or minus one standard deviation.

<p>Values correspond to absorbance at 405 nm. − represents no detected absorption. + represents at least two times the average absorbance of buffer control.</p